Antibodies to human Ependymin

ABSTRACT

The present invention relates to a novel human Ependymin protein which is a member of the ependymin family. In particular, isolated nucleic acid molecules are provided encoding the human Ependymin protein. Ependymin polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of Ependymin activity. Also provided are diagnostic methods for detecting nervous system-related disorders and therapeutic methods for treating nervous system-related disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/187,904, filed Jul. 3, 2002, (now U.S. Pat. No. 6,683,161, issuedJan. 27, 2004), which is a divisional of U.S. application Ser. No.09/229,583, filed Jan. 13, 1999, (now U.S. Pat. No. 6,489,138, issuedDec. 3, 2002), which claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Nos. 60/071,330, filed on Jan. 14, 1998, and60/075,278, filed on Feb. 19, 1998, each of which is hereby incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a novel human gene encoding apolypeptide which is a member of the Ependymin family. Morespecifically, isolated nucleic acid molecules are provided encoding ahuman polypeptide named Ependymin. Ependymin polypeptides are alsoprovided, as are vectors, host cells and recombinant methods forproducing the same. Also provided are diagnostic methods for detectingdisorders related to the nervous system, and therapeutic methods fortreating such disorders.

The invention further relates to screening methods for identifyingagonists and antagonists of Ependymin activity.

BACKGROUND OF THE INVENTION

Within the last several years, a number of ependymins have beenmolecularly cloned from a variety of teleost fish including Oncorhynchusmykiss (rainbow trout; Muller-Schmid, A., et al., Gene 118:189-196(1992)), Salmo salar (Atlantic salmon; Muller-Schmid, A., et al., Gene118:189-196 (1992)), Esox lucius (pike; Muller-Schmid, A., et al., J.Molec. Evol. 36:578-585 (1993)), Carassius auratus (goldfish;Konigstorfer, A., et al., J. Neurochem. 52:310-312 (1989); Konigstorfer,A., et al., J. Biol. Chem. 264:13689-13692 (1989)), Brachydanio rerio(zebrafish; Sterrer, S., et al., Neurosci. 37:277-284 (1990)), andClupea harengus (herring, Muller-Schmid, et al., J. Molec. Evol.36:578-585 (1993)). The ependymins produced by these organisms aresynthesized as precursors which contain N-terminal, hydrophobic signalsequences. Each of these molecules contains multiple N-linkedglycosylation sites, only some of which are conserved between species(Schmidt, R. and Shashoua, V. E. J. Neurochem. 36:1368-1377 (1981);Schmidt, R. and Shashoua, V. E. J. Neurochem. 40:652-660 (1983); Ganb,B. and Hoffman, W. Eur. J. Biochem. 217:275-280 (1993)). The precursorependymins range in apparent molecular mass from 23.7 to 24.5 kDa, whilethe secreted mature forms of these molecules are typically 21.6-22.3 kDain size.

The piscine ependymins characterized thus far may be categorizedaccording to the number of cysteine residues present in the maturepolypeptide. Mature salmoniform (O. mykiss, S. salar, and E. lucius)ependymin polypeptides contain only four cysteine residues, whereasmature cypriniform (C. auratus and B. rerio) and clupeiform (C.harengus) ependymin polypeptides contain five and six cysteine residues,respectively (Hoffmann, W. Int. J. Biochem. 26:607-619 (1994)).Correspondingly, disulfide-linked dimerization of the salmoniformependymin polypeptides is not observed after non-reducing SDS-PAGE.However, cypriniform and clupeiform ependymins are observed asdisulfide-linked dimers under non-reducing conditions. It is speculatedthat the dimerization occurs via the cysteine residue conserved onlybetween the salmoniform ependymins (this cysteine residue aligns withthe lysine residue at location 133 of human ependymin of the presentinvention as shown in SEQ ID NO:2).

Several lines of evidence have provided the basis for an understandingof the functional role(s) of the ependymins. Ca²⁺-binding has beendemonstrated for at least goldfish and rainbow trout ependymins(Schmidt, R. and Makiola, E. Neuro. Chem. (Life Sci. Adv.) 10:161-171(1991); Ganb, B. and Hoffman, supra). Further, ependymins are theprimary cerebrospinal fluid component in a number of teleost fish(Schmidt, R. and Lapp, H. Neurochem. Int. 10:383-390 1987). Finally,roughly two-thirds of Ca2+ in the CSF of rainbow trout is protein-bound(Ganb, B. and Hoffman, supra). As a result, it is thought thatependymins may function in Ca²⁺ homeostasis of the teleost piscine brain(Hoffman, W., supra).

In situ hybridization analyses have shown that ependymins are apparentlysynthesized exclusively in miningeal fibroblasts of the mininx (alsotermed the endomeninx of leptomeninx) of teleost fish (Konigstorfer, A.,et al., Cell Tissue Res. 261:59-64 (1990)). Ependymins have also beenfound to associate with collagen fibrils of the extracellular matrix(ECM; Schwarz, H., et al. Cell Tissue Res. 273:417-425 (1993)), and,further, have the capacity to serve as a substrate for outgrowingretinal axons (Schmidt, J. T., et al., J. Neurobiol. 22:40-54 (1991)).

An additional role for ependymins has been identified in the field oflearning and memory. Using an experimental approach in which goldfishlearn to swim to a specific compartment of its environment to avoid anelectric shock, investigators have determined that the amount of unboundor unincorporated extracellular ependymins decreases after learning(Piront, M.-L., and Schmidt, R. Brain Res. 442:53-62 (1988); Schmidt, R.J. Neurochem. 48:1870-1878 (1987)). Further, blockage of functionalependymin molecules, either with antibodies or antisensepolynucleotides, resulted in the reversible inability of theexperimental animal to remember the task which it had learned. Removalof the inhibitory substance then resulted in a reappearance of thelearned ability (Schmidt, R. J. supra; Shashoua, V. E. and Moore, M. E.Brain Res. 148:441-449 (1978)).

Thus, there is a need for polypeptides that function as neurotrophicfactors in the regeneration of the optic and other nerves and inlong-term memory consolidation, since disturbances of such regulationmay be involved in disorders relating to the complex molecular andcellular process regulating neuronal and nervous system function. Suchdisorders may include Parkinson's disease, Alzheimer's disease,amyotrophic lateral sclerosis, pain, stroke, depression, anxiety,epilepsy, and other neurological and psychiatric disorders. Therefore,there is a need for identification and characterization of such humanpolypeptides which can play a role in detecting, preventing,ameliorating or correcting such disorders.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding at least a portion of the Ependyminpolypeptide having the complete amino acid sequence shown in SEQ ID NO:2or the complete amino acid sequence encoded by the cDNA clone depositedas plasmid DNA as ATCC Deposit Number 209464 on Nov. 14, 1997. Thenucleotide sequence determined by sequencing the deposited Ependyminclone, which is shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1), contains anopen reading frame encoding a complete polypeptide of 224 amino acidresidues, including an initiation codon encoding an N-terminalmethionine at nucleotide positions 296-298, and a predicted molecularweight of about 25.4 kDa. Nucleic acid molecules of the inventioninclude those encoding the complete amino acid sequence excepting theN-terminal methionine shown in SEQ ID NO:2, or the complete amino acidsequence excepting the N-terminal methionine encoded by the cDNA clonein ATCC Deposit Number 209464, which molecules also can encodeadditional amino acids fused to the N-terminus of the Ependymin aminoacid sequence.

The encoded polypeptide has a predicted leader sequence of 37 aminoacids underlined in FIGS. 1A, 1B, and 1C; and the amino acid sequence ofthe predicted mature Ependymin protein is also shown in FIGS. 1A, 1B,and 1C, as amino acid residues 38-224 and as residues 1-187 in SEQ IDNO:2.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the Ependymin polypeptide having the complete amino acidsequence in SEQ ID NO:2 (i.e., positions −37 to 187 of SEQ ID NO:2); (b)a nucleotide sequence encoding the Ependymin polypeptide having thecomplete amino acid sequence in SEQ ID NO:2 excepting the N-terminalmethionine (i.e., positions −36 to 187 of SEQ ID NO:2); (c) a nucleotidesequence encoding the predicted mature Ependymin polypeptide having theamino acid sequence at positions 1 to 187 in SEQ ID NO:2; (d) anucleotide sequence encoding the Ependymin polypeptide having thecomplete amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 209464; (e) a nucleotide sequence encoding the Ependyminpolypeptide having the complete amino acid sequence excepting theN-terminal methionine encoded by the cDNA clone contained in ATCCDeposit No. 209464; (f) a nucleotide sequence encoding the matureEpendymin polypeptide having the amino acid sequence encoded by the cDNAclone contained in ATCC Deposit No. 209464; and (g) a nucleotidesequence complementary to any of the nucleotide sequences in (a), (b),(c), (d), (e) or (f), above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical, to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f) or (g), above, or a polynucleotide which hybridizes understringent hybridization conditions to a polynucleotide in (a), (b), (c),(d), (e), (f) or (g), above. This polynucleotide which hybridizes doesnot hybridize under stringent hybridization conditions to apolynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues.

An additional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of a Ependyminpolypeptide having an amino acid sequence in (a), (b), (c), (d), (e) or(f), above. A further nucleic acid embodiment of the invention relatesto an isolated nucleic acid molecule comprising a polynucleotide whichencodes the amino acid sequence of a Ependymin polypeptide having anamino acid sequence which contains at least one conservative amino acidsubstitution, but not more than 50 conservative amino acidsubstitutions, even more preferably, not more than 40 conservative aminoacid substitutions, still more preferably, not more than 30 conservativeamino acid substitutions, and still even more preferably, not more than20 conservative amino acid substitutions. Of course, in order ofever-increasing preference, it is highly preferable for a polynucleotidewhich encodes the amino acid sequence of a Ependymin polypeptide to havean amino acid sequence which contains not more than 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 conservative amino acid substitutions.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofEpendymin polypeptides or peptides by recombinant techniques.

The invention further provides an isolated Ependymin polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of the full-length Ependymin polypeptidehaving the complete amino acid sequence shown in SEQ ID NO:2 (i.e.,positions −37 to 187 of SEQ ID NO:2); (b) the amino acid sequence of thefull-length Ependymin polypeptide having the complete amino acidsequence shown in SEQ ID NO:2 excepting the N-terminal methionine (i.e.,positions −36 to 187 of SEQ ID NO:2); (c) the amino acid sequence of thepredicted mature Ependymin polypeptide having the amino acid sequence atpositions 1 to 187 in SEQ ID NO:2; (d) the complete amino acid sequenceencoded by the cDNA clone contained in the ATCC Deposit No. 209464; (e)the complete amino acid sequence excepting the N-terminal methionineencoded by the cDNA clone contained in the ATCC Deposit No. 209464; and(f) the complete amino acid sequence of the predicted mature Ependyminpolypeptide encoded by the cDNA clone contained in the ATCC Deposit No.209464. The polypeptides of the present invention also includepolypeptides having an amino acid sequence at least 80% identical, morepreferably at least 90% identical, and still more preferably 95%, 96%,97%, 98% or 99% identical to those described in (a), (b), (c), (d), (e)or (f), above, as well as polypeptides having an amino acid sequencewith at least 90% similarity, and more preferably at least 95%similarity, to those above.

An additional embodiment of this aspect of the invention relates to apeptide or polypeptide which comprises the amino acid sequence of anepitope-bearing portion of a Ependymin polypeptide having an amino acidsequence described in (a), (b), (c), (d), (e) or (f), above. Peptides orpolypeptides having the amino acid sequence of an epitope-bearingportion of a Ependymin polypeptide of the invention include portions ofsuch polypeptides with at least six or seven, preferably at least nine,and more preferably at least about 30 amino acids to about 50 aminoacids, although epitope-bearing polypeptides of any length up to andincluding the entire amino acid sequence of a polypeptide of theinvention described above also are included in the invention.

A further embodiment of the invention relates to a peptide orpolypeptide which comprises the amino acid sequence of a Ependyminpolypeptide having an amino acid sequence which contains at least oneconservative amino acid substitution, but not more than 50 conservativeamino acid substitutions, even more preferably, not more than 40conservative amino acid substitutions, still more preferably, not morethan 30 conservative amino acid substitutions, and still even morepreferably, not more than 20 conservative amino acid substitutions. Ofcourse, in order of ever-increasing preference, it is highly preferablefor a peptide or polypeptide to have an amino acid sequence whichcomprises the amino acid sequence of a Ependymin polypeptide, whichcontains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1conservative amino acid substitutions.

In another embodiment, the invention provides an isolated antibody thatbinds specifically to a Ependymin polypeptide having an amino acidsequence described in (a), (b), (c), (d), (e) or (f) above. Theinvention further provides methods for isolating antibodies that bindspecifically to a Ependymin polypeptide having an amino acid sequence asdescribed herein. Such antibodies are useful diagnostically ortherapeutically as described below.

The invention also provides for pharmaceutical compositions comprisingEpendymin polypeptides, particularly human Ependymin polypeptides, whichmay be employed, for instance, to treat Parkinson's disease, Alzheimer'sdisease, amyotrophic lateral sclerosis, pain, stroke, depression,anxiety, epilepsy, and other neurological and psychiatric disorders.Methods of treating individuals in need of Ependymin polypeptides arealso provided.

The invention further provides compositions comprising a Ependyminpolynucleotide or an Ependymin polypeptide for administration to cellsin vitro, to cells ex vivo and to cells in vivo, or to a multicellularorganism. In certain particularly preferred embodiments of this aspectof the invention, the compositions comprise a Ependymin polynucleotidefor expression of a Ependymin polypeptide in a host organism fortreatment of disease. Particularly preferred in this regard isexpression in a human patient for treatment of a dysfunction associatedwith aberrant endogenous activity of a Ependymin

In another aspect, a screening assay for agonists and antagonists isprovided which involves determining the effect a candidate compound hason Ependymin binding to a receptor. In particular, the method involvescontacting the receptor with a Ependymin polypeptide and a candidatecompound and determining whether Ependymin polypeptide binding to thereceptor is increased or decreased due to the presence of the candidatecompound. In this assay, an increase in binding of Ependymin over thestandard binding indicates that the candidate compound is an agonist ofEpendymin binding activity and a decrease in Ependymin binding comparedto the standard indicates that the compound is an antagonist ofEpendymin binding activity.

In yet another aspect, the Ependymin may bind to a cell surface proteinwhich also function as a viral receptor or coreceptor. Thus, Ependymin,or agonists or antagonists thereof, may be used to regulate viralinfectivity at the level of viral binding or interaction with theEpendymin receptor or coreceptor or during the process of viralinternalization or entry into the cell.

It has been discovered that Ependymin is expressed not only in primarydendritic cells, but also in the KMH2 cell line, placenta, fetal andadult liver, spinal cord, osteoclastoma, cerebellum, synovialfibroblasts, 12 week old early stage human embryo, adrenal gland tumor,whole brain, Hodgkin's Lymphoma tissue, macrophages, HEL cell line, andchondrosarcoma. Therefore, nucleic acids of the invention are useful ashybridization probes for differential identification of the tissue(s) orcell type(s) present in a biological sample. Similarly, polypeptides andantibodies directed to those polypeptides are useful to provideimmunological probes for differential identification of the tissue(s) orcell type(s). In addition, for a number of disorders of the abovetissues or cells, particularly of the nervous system, significantlyhigher or lower levels of Ependymin gene expression may be detected incertain tissues (e.g., cancerous and wounded tissues) or bodily fluids(e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken froman individual having such a disorder, relative to a “standard” Ependymingene expression level, i.e., the Ependymin expression level in healthytissue from an individual not having the nervous system disorder. Thus,the invention provides a diagnostic method useful during diagnosis ofsuch a disorder, which involves: (a) assaying Ependymin gene expressionlevel in cells or body fluid of an individual; (b) comparing theEpendymin gene expression level with a standard Ependymin geneexpression level, whereby an increase or decrease in the assayedEpendymin gene expression level compared to the standard expressionlevel is indicative of disorder in the nervous system.

An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of Ependyminactivity in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of an isolatedEpendymin polypeptide of the invention or an agonist thereof.

A still further aspect of the invention is related to a method fortreating an individual in need of a decreased level of Ependyminactivity in the body comprising, administering to such an individual acomposition comprising a therapeutically effective amount of anEpendymin antagonist. Preferred antagonists for use in the presentinvention are Ependymin-specific antibodies.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, and 1C show the nucleotide sequence (SEQ ID NO:1) anddeduced amino acid sequence (SEQ ID NO:2) of Ependymin. The predictedleader sequence of about 37 amino acids is underlined. Note that themethionine residue at the beginning of the leader sequence in FIGS. 1A,1B, and 1C is shown in position number (positive) 1, whereas the leaderpositions in the corresponding sequence of SEQ ID NO:2 are designatedwith negative position numbers. Thus, the leader sequence positions 1 to37 in FIGS. 1A, 1B, and 1C correspond to positions −37 to −1 in SEQ IDNO:2.

Two potential asparagine-linked glycosylation sites are marked in theamino acid sequence of Ependymin. The sites are asparagine-130 andasparagine-182 in FIGS. 1A, 1B, and 1C (asparagine-93 and asparagine-145in SEQ ID NO:2), and are labeled with the bold pound symbol (#) abovethe nucleotide sequence coupled with a bolded one letter abbreviationfor the asparagine (N) in the amino acid sequence in FIGS. 1A, 1B, and1C; that is, the actual asparagine residues which are potentiallyglycosylated is bolded in FIGS. 1A, 1B, and 1C.

A polyadenylation signal sequence is present in the 3′ untranslatedregion of the nucleotide sequence of Ependymin of the present invention.The polyadenylation signal sequence is delineated by a double underlinein the Ependymin sequence shown in FIGS. 1A, 1B, and 1C.

Regions of high identity between Human Ependymin of the presentinvention and several previously identified piscine ependymins (analignment of these sequences is presented in FIG. 2) are delineated inFIGS. 1A, 1B, and 1C with a double underline. These regions are notlimiting and are labeled as Conserved Domain (CD)-II, CD-III, CD-IV,CD-V, CD-VI, CD-VII, CD-IX, and CD-X in FIGS. 1A, 1B, and 1C.

FIG. 2 shows the regions of identity between the amino acid sequences ofthe Ependymin protein and translation product of the Zebrafish mRNA forEpendymin (SEQ ID NO:6), determined by the computer program Bestfit(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711) using the default parameters.

FIGS. 3A and 3B are a multiple sequence alignment illustrating acomparison of the amino acid sequences of the novel human ependymin ofthe present invention (SEQ ID NO:2) and five previously describedependymin-like molecules. Conserved residues are highlighted in black.The ependymin homologues shown in the figure are goldfish ependymin II(SEQ ID NO:3; GenBank Accession No. J04986), rainbow trout ependymin II(SEQ ID NO:4; GenBank Accession No. M93698), common carp ependymin (SEQID NO:5; GenBank Accession No. U00432), zebrafish ependymin (SEQ IDNO:6; GenBank Accession No. X52502), and Atlantic herring ependymin (SEQID NO:7; GenBank Accession No. L09065).

FIG. 4 shows an analysis of the Ependymin amino acid sequence. Alpha,beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index or Jameson-Wolf” graph,the positive peaks indicate locations of the highly antigenic regions ofthe Ependymin protein, i.e., regions from which epitope-bearing peptidesof the invention can be obtained.

The data presented in FIG. 4 are also represented in tabular form inTable I. The columns are labeled with the headings “Res”, “Position”,and Roman Numerals I-XIV. The column headings refer to the followingfeatures of the amino acid sequence presented in FIG. 4 and Table I:“Res”: amino acid residue of FIGS. 1A, 1B, and 1C (which is theidentical sequence shown in SEQ ID NO:2, with the exception that theresidues are numbered 1-224 in FIGS. 1A, 1B, and 1C and −37 through 187in SEQ ID NO:2); “Position”: position of the corresponding residuewithin FIGS. 1A, 1B, and 1C (which is the identical sequence shown inSEQ ID NO:2, with the exception that the residues are numbered 1-224 inFIGS. 1A, 1B, and 1C and −37 through 187 in SEQ ID NO:2); I: Alpha,Regions—Garnier-Robson; II: Alpha, Regions—Chou-Fasman; III: Beta,Regions—Garnier-Robson; IV: Beta, Regions—Chou-Fasman; V: Turn,Regions—Garnier-Robson; VI: Turn, Regions—Chou-Fasman; VII: Coil,Regions—Garnier-Robson; VIII: Hydrophilicity Plot—Kyte-Doolittle; IX:Hydrophobicity Plot—Hopp-Woods; X: Alpha, Amphipathic Regions—Eisenberg;XI: Beta, Amphipathic Regions—Eisenberg; XII: FlexibleRegions—Karplus-Schulz; XIII: Antigenic Index—Jameson-Wolf; and XIV:Surface Probability Plot—Emini.

DETAILED DESCRIPTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a Ependymin polypeptide having theamino acid sequence shown in SEQ ID NO:2, which was determined bysequencing a cloned cDNA. The nucleotide sequence shown in FIGS. 1A, 1B,and 1C (SEQ ID NO:1) was obtained by sequencing the HDPIE88 clone, whichwas deposited on Nov. 14, 1997 at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209, and givenaccession number ATCC 209464. The deposited clone is contained in thepBluescript SK(−) plasmid (Stratagene, La Jolla, Calif.).

The Ependymin protein of the present invention shares sequence homologywith the translation products of ependymins from a number of teleostfish including the Zebrafish mRNA for ependymin (FIG. 2; SEQ ID NO:6).Zebrafish ependymin, and other ependymins, are thought to be involved inthe regulation of extracellular Ca ²⁺-binding. In fact, as thepredominant cerebrospinal fluid constituents in many teleost fish,ependymins are believed to be antiadhesive extracellular matrixglycoproteins which function in the processes of cell contact andregeneration.

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc., FosterCity, Calif.), and all amino acid sequences of polypeptides encoded byDNA molecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

By “nucleotide sequence” of a nucleic acid molecule or polynucleotide isintended, for a DNA molecule or polynucleotide, a sequence ofdeoxyribonucleotides, and for an RNA molecule or polynucleotide, thecorresponding sequence of ribonucleotides (A, G, C and U), where eachthymidine deoxyribonucleotide (T) in the specified deoxyribonucleotidesequence is replaced by the ribonucleotide uridine (U).

Using the information provided herein, such as the nucleotide sequencein FIGS. 1A, 1B, and 1C (SEQ ID NO:1), a nucleic acid molecule of thepresent invention encoding a Ependymin polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 1A, 1B, and 1C (SEQ IDNO:1) was discovered in a cDNA library derived from primary dendriticcells.

Additional clones of the same gene were also identified in cDNAlibraries from the following tissues: the KMH2 cell line, placenta,fetal and adult liver, spinal cord, osteoclastoma, cerebellum, synovialfibroblasts, 12 week old early stage human embryo, adrenal gland tumor,whole brain, Hodgkin's Lymphoma tissue, macrophages, HEL cell line, andchondrosarcoma.

The determined nucleotide sequence of the Ependymin cDNA of FIGS. 1A,1B, and 1C (SEQ ID NO:1) contains an open reading frame encoding aprotein of 224 amino acid residues, with an initiation codon atnucleotide positions 296-298 of the nucleotide sequence in FIGS. 1A, 1B,and 1C (SEQ ID NO:1), and a deduced molecular weight of about 25.4 kDa.The amino acid sequence of the Ependymin protein shown in SEQ ID NO:2 isabout 22.5% identical to Zebrafish mRNA for ependymin (FIG. 2; Sterrer,S., et al., Neurosci. 37:277-284 (1990); GenBank Accession No. X52502).

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above, the actual complete Ependyminpolypeptide encoded by the deposited cDNA, which comprises about 224amino acids, may be somewhat longer or shorter. It will further beappreciated that, depending on the analytical criteria used foridentifying various functional domains, the exact “address” of thesignal peptide and mature regions of the Ependymin polypeptide maydiffer slightly from the predicted positions above. For example, theexact location of the Ependymin signal peptide in SEQ ID NO:2 may varyslightly (e.g., the address may “shift” by about 1 to about 15 residues,more likely about 1 to about 10 residues, and even more likely about 1to about 5 residues) depending on the criteria used to define thedomain. In this case, the end of the signal peptide and the beginning ofthe mature Ependymin molecule were predicted using the Human GenomeSciences, Inc. (HGSI) SignalP computer algorithm (Pedersen, A. G. andNielsen, H. ISMB 5:226-233 (1997)). One of skill in the art will realizethat another widely accepted computer algorithm used to predictpotential sites of polypeptide cleavage, PSORT, will predict thecleavage of an N-terminal signal peptide from the Ependymin polypeptideat a point slightly different from that predicted by the HGSI SignalPalgorithm. In any event, as discussed further below, the inventionfurther provides polypeptides having various residues deleted from theN-terminus of the complete polypeptide, including polypeptides lackingone or more amino acids from the N-terminus of the mature polypeptidedescribed herein.

Leader and Mature Sequences

The amino acid sequence of the complete Ependymin protein includes aleader sequence and a mature protein, as shown in SEQ ID NO:2. More inparticular, the present invention provides nucleic acid moleculesencoding a mature form of the Ependymin protein. Thus, according to thesignal hypothesis, once export of the growing protein chain across therough endoplasmic reticulum has been initiated, proteins secreted bymammalian cells have a signal or secretory leader sequence which iscleaved from the complete polypeptide to produce a secreted “mature”form of the protein. Most mammalian cells and even insect cells cleavesecreted proteins with the same specificity. However, in some cases,cleavage of a secreted protein is not entirely uniform, which results intwo or more mature species of the protein. Further, it has long beenknown that the cleavage specificity of a secreted protein is ultimatelydetermined by the primary structure of the complete protein, that is, itis inherent in the amino acid sequence of the polypeptide. Therefore,the present invention provides a nucleotide sequence encoding the matureEpendymin polypeptide having the amino acid sequence encoded by the cDNAclone contained in ATCC Deposit No. 209464. By the “mature Ependyminpolypeptide having the amino acid sequence encoded by the cDNA clone inATCC Deposit No. 209464” is meant the mature form(s) of the Ependyminprotein produced by expression in a mammalian cell (e.g., COS cells, asdescribed below) of the complete open reading frame encoded by the humanDNA sequence of the deposited clone.

In addition, methods for predicting whether a protein has a secretoryleader as well as the cleavage point for that leader sequence areavailable. For instance, the method of McGeoch (Virus Res. 3:271-286(1985)) uses the information from a short N-terminal charged region anda subsequent uncharged region of the complete (uncleaved) protein. Themethod of von Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) uses theinformation from the residues surrounding the cleavage site, typicallyresidues −13 to +2 where +1 indicates the amino terminus of the matureprotein. The accuracy of predicting the cleavage points of knownmammalian secretory proteins for each of these methods is in the rangeof 75-80% (von Heinje, supra). However, the two methods do not alwaysproduce the same predicted cleavage point(s) for a given protein.

In the present case, the deduced amino acid sequence of the completeEpendymin polypeptide was analyzed by the HGSI SignalP computeralgorithm (Pedersen, A. G. and Nielsen, H. ISMB 5:226-233 (1997)). Usingthis computer analysis tool, a likely site of signal peptide cleavagewas predicted between amino acid residues 37 and 38 of the, Ependyminsequence (SEQ ID NO:2). However, the deduced amino acid sequence of thecomplete Ependymin polypeptide was also analyzed by a computer programdesignated “PSORT”, available from Dr. Kenta Nakai of the Institute forChemical Research, Kyoto University (Nakai, K. and Kanehisa, M. Genomics14:897-911 (1992)), which is an expert system for predicting thecellular location of a protein based on the amino acid sequence. As partof this computational prediction of localization, the methods of McGeochand von Heinje are incorporated. Thus, the computation analysis abovepredicted a single cleavage site within the complete amino acid sequenceshown in SEQ ID NO:2.

As one of ordinary skill would appreciate from the above discussions,due to the possibilities of sequencing errors as well as the variabilityof cleavage sites in different known proteins, the mature Ependyminpolypeptide encoded by the deposited cDNA is expected to consist ofabout 187 amino acids (presumably residues 1 to 187 of SEQ ID NO:2)based on analysis of the Ependymin amino acid sequence using the SignalPcomputer algorithm, but may consist of any number of amino acids in therange of about 187 to 200 amino acids (the mature polypeptide ispredicted to be 200 amino acids using the PSORT computer algorithm).Further, the actual leader sequence(s) of this protein is expected to be37 amino acids (presumably residues −37 through −1 of SEQ ID NO:2) basedon analysis of the Ependymin amino acid sequence using the SignalPcomputer algorithm, but may consist of any number of amino acids in therange of 24-37 amino acids (the signal peptide is predicted to be 24amino acids using the PSORT computer algorithm).

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) with an initiationcodon at positions 288-290 of the nucleotide sequence shown in FIGS. 1A,1B, and 1C (SEQ ID NO:1).

Also included are DNA molecules comprising the coding sequence for thepredicted mature Ependymin protein shown at positions 1-187 of SEQ IDNO:2.

In addition, isolated nucleic acid molecules of the invention includeDNA molecules which comprise a sequence substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encode the Ependymin protein. Of course, the genetic codeand species-specific codon preferences are well known in the art. Thus,it would be routine for one skilled in the art to generate thedegenerate variants described above, for instance, to optimize codonexpression for a particular host (e.g., change codons in the human mRNAto those preferred by a bacterial host such as E. coli).

In another aspect, the invention provides isolated nucleic acidmolecules encoding the Ependymin polypeptide having an amino acidsequence encoded by the cDNA clone contained in the plasmid deposited asATCC Deposit No. 209464 on Nov. 14, 1997.

Preferably, this nucleic acid molecule will encode the maturepolypeptide encoded by the above-described deposited cDNA clone.

The invention further provides an isolated nucleic acid molecule havingthe nucleotide sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) orthe nucleotide sequence of the Ependymin cDNA contained in theabove-described deposited clone, or a nucleic acid molecule having asequence complementary to one of the above sequences. Such isolatedmolecules, particularly DNA molecules, are useful as probes for genemapping, by in situ hybridization with chromosomes, and for detectingexpression of the Ependymin gene in human tissue, for instance, byNorthern blot analysis.

The present invention is further directed to nucleic acid moleculesencoding portions of the nucleotide sequences described herein as wellas to fragments of the isolated nucleic acid molecules described herein.In particular, the invention provides a polynucleotide having anucleotide sequence representing the portion of SEQ ID NO:1 whichconsists of positions 1-967 of SEQ ID NO:1.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:1 whichhave been determined from the following related cDNA clones: HATBS80R(SEQ ID NO:15); HSRAN11R (SEQ ID NO:16); HCECE56R (SEQ ID NO:17);HSNBF20R (SEQ ID NO:18); HPMDJ94R (SEQ ID NO:19); and HE2FK31R (SEQ IDNO:20).

Further, the invention includes a polynucleotide comprising any portionof at least about 30 nucleotides, preferably at least about 50nucleotides, of SEQ ID NO:1 from residue 1 to 2505.

More generally, by a fragment of an isolated nucleic acid moleculehaving the nucleotide sequence of the deposited cDNA or the nucleotidesequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) is intendedfragments at least about 15 nt, and more preferably at least about 20nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50-300 nt in length are also useful according to the presentinvention as are fragments corresponding to most, if not all, of thenucleotide sequence of the deposited cDNA or as shown in FIGS. 1A, 1B,and 1C (SEQ ID NO:1). By a fragment at least 20 nt in length, forexample, is intended fragments which include 20 or more contiguous basesfrom the nucleotide sequence of the deposited cDNA or the nucleotidesequence as shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1). Preferrednucleic acid fragments of the present invention include nucleic acidmolecules encoding epitope-bearing portions of the Ependymin polypeptideas identified in FIGS. 3A and 3B and described in more detail below.

Preferred nucleic acid fragments of the present invention also includenucleic acid molecules encoding one or more of the following domains ofHuman Ependymin: amino acid residues −20 through −10 of SEQ ID NO:2;amino acid residues 1 through 10 of SEQ ID NO:2; amino acid residues 31through 40 of SEQ ID NO:2; amino acid residues 64 through 70 of SEQ IDNO:2; amino acid residues 76 through 82 of SEQ ID NO:2; amino acidresidues 88 through 105 of SEQ ID NO:2; amino acid residues 106 through117 of SEQ ID NO:2; amino acid residues 129 through 143 of SEQ ID NO:2;amino acid residues 150 through 156 of SEQ ID NO:2; and amino acidresidues 162 through 184 of SEQ ID NO:2. These domains are representedas conserved domains CD-I through CD-X in FIGS. 1A, 1B, and 1C.

In specific embodiments, the polynucleotide fragments of the inventionencode antigenic regions. Non-limiting examples of antigenicpolypeptides or peptides that can be used to generate Ependymin-specificantibodies include: a polypeptide comprising amino acid residues fromabout Ala-1 to about Gln-9 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about Pro-8 to about Val-16 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about Gln-19 to aboutArg-27 in SEQ ID NO:2; a polypeptide comprising amino acid residues fromabout Ile-69 to about Ser-77 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about Asp-86 to about Glu-107 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about Glu-113 to aboutTyr-123 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about Thr-131 to about Gln-139 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about Leu-159 to about Phe-167 inSEQ ID NO:2; and a polypeptide comprising amino acid residues from aboutLeu-178 to about Ser-186 in SEQ ID NO:2.

In additional embodiments, the polynucleotides of the invention encodefunctional attributes of Human Ependymin. Preferred embodiments of theinvention in this regard include fragments that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheetforming regions (“beta-regions”), turn and turn-forming regions(“turn-regions”), coil and coil-forming regions (“coil-regions”),hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions andhigh antigenic index regions of Human Ependymin.

The data representing the structural or functional attributes of HumanEpendymin set forth in FIG. 4 and/or Table I, as described above, wasgenerated using the various modules and algorithms of the DNA*STAR seton default parameters. In a preferred embodiment, the data presented incolumns VIII, IX, XIII, and XIV of Table I can be used to determineregions of Human Ependymin which exhibit a high degree of potential forantigenicity. Regions of high antigenicity are determined from the datapresented in columns VIII, IX, XIII, and/or IV by choosing values whichrepresent regions of the polypeptide which are likely to be exposed onthe surface of the polypeptide in an environment in which antigenrecognition may occur in the process of initiation of an immuneresponse.

Certain preferred regions in these regards are set out in FIG. 4, butmay, as shown in Table I, be represented or identified by using tabularrepresentations of the data presented in FIG. 4. The DNA*STAR computeralgorithm used to generate FIG. 4 (set on the original defaultparameters) was used to present the data in FIG. 4 in a tabular format(See Table I). The tabular format of the data in FIG. 4 may be used toeasily determine specific boundaries of a preferred region.

The above-mentioned preferred regions set out in FIG. 4 and in Table Iinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS. 1A,1B, and 1C. As set out in FIG. 4 and in Table I, such preferred regionsinclude Garnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions,Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenbergalpha- and beta-amphipathic regions, Karplus-Schulz flexible regions,Emini surface-forming regions and Jameson-Wolf regions of high antigenicindex.

TABLE I Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met1 . . B . . T . 0.31 −0.24 * * . 1.07 1.11 Pro 2 . . . . . T C 0.49−0.17 . * . 1.34 0.88 Gly 3 . . . . T T . 0.07 −0.17 * * . 1.91 1.06 Arg4 . . B . . T . 0.57 0.09 * * . 0.98 0.89 Ala 5 . . B . . . . 0.64−0.53 * * F 2.20 1.12 Pro 6 . . B B . . . 0.39 −0.47 * * F 1.48 1.63 Leu7 . . B B . . . 0.39 −0.26 * * . 0.96 0.62 Arg 8 . . B B . . . 0.390.17 * * F 0.29 0.95 Thr 9 . . B B . . . −0.31 0.10 * * F 0.07 0.61 Val10 . . B . . T . −0.53 0.17 * * F 0.25 0.74 Pro 11 . . B . . T . −0.670.17 * . F 0.25 0.31 Gly 12 . . B . . T . −0.44 0.60 * . F −0.05 0.21Ala 13 . . B . . T . −0.84 0.61 * . . −0.20 0.29 Leu 14 . A B B . . .−1.34 0.89 . . . −0.60 0.20 Gly 15 . A B B . . . −1.30 1.14 . . . −0.600.17 Ala 16 . A B B . . . −1.43 1.40 . . . −0.60 0.14 Trp 17 . A B B . .. −1.43 1.33 . . . −0.60 0.16 Leu 18 . A B B . . . −1.66 1.07 . . .−0.60 0.16 Leu 19 . A B B . . . −1.13 1.33 . . . −0.60 0.13 Gly 20 . A .B T . . −1.38 1.74 . . . −0.20 0.13 Gly 21 . . . B . . C −1.08 1.33 . .. −0.40 0.16 Leu 22 . . . B T . . −1.10 1.56 . . . −0.20 0.21 Trp 23 . .. B T . . −1.10 1.36 . . . −0.20 0.30 Ala 24 . . B B . . . −0.96 1.61 .. . −0.60 0.25 Trp 25 . . B B . . . −0.96 1.76 . . . −0.60 0.16 Thr 26 .. B B . . . −1.42 1.50 . . . −0.60 0.15 Leu 27 . . B B . . . −1.28 1.27. . . −0.60 0.13 Cys 28 . . . B T . . −1.29 1.34 . . . −0.20 0.06 Gly 29. . . B T . . −1.51 0.81 . . . −0.20 0.06 Leu 30 . . . B T . . −1.571.01 . . . −0.20 0.06 Cys 31 . . B . . T . −1.84 0.76 . . . −0.20 0.11Ser 32 . . B . . T . −1.89 0.69 . . . −0.20 0.11 Leu 33 . . B . . T .−1.57 0.90 . . . −0.20 0.10 Gly 34 . . B . . T . −1.81 0.64 . . . −0.200.19 Ala 35 . . B B . . . −1.21 0.57 . . . −0.60 0.14 Val 36 . . B B . .. −0.43 0.61 . . . −0.60 0.26 Gly 37 . . B B . . . −0.34 −0.07 . . .0.58 0.52 Ala 38 . . B . . . . −0.20 −0.07 . . F 1.21 0.80 Pro 39 . . B. . . . 0.14 −0.00 * . F 1.49 0.58 Arg 40 . . . . . T C 0.14 −0.24 * . F2.32 1.01 Pro 41 . . . . T T . 0.79 −0.17 . . F 2.80 1.01 Cys 42 . . . .T T . 1.13 −0.24 * . F 2.52 1.01 Gln 43 . . B . . T . 1.72 −0.27 * . F1.69 0.89 Ala 44 . . B . . . . 1.64 0.13 * * F 0.61 1.00 Pro 45 . . . .. . C 1.53 0.61 * * F 0.58 1.96 Gln 46 . . . . T . . 1.40 0.04 * * F1.00 1.96 Gln 47 . . . . T . . 2.18 0.07 . * F 1.20 1.92 Trp 48 . . . .T . . 2.18 −0.43 . * F 2.00 2.43 Glu 49 . . . . . . C 1.91 −0.46 . * F2.00 2.43 Gly 50 . . . B T . . 1.52 −0.21 . * F 1.80 1.04 Arg 51 . . . BT . . 1.28 −0.00 . * F 1.45 0.98 Gln 52 . . B B . . . 1.28 −0.16 . * .0.70 0.89 Val 53 . . B B . . . 1.57 0.24 . * . 0.05 1.55 Met 54 . . B B. . . 1.27 0.21 . . . −0.15 1.37 Tyr 55 . . B B . . . 1.31 0.60 * . .−0.45 1.06 Gln 56 . . B B . . . 0.86 0.59 * . F 0.04 1.91 Gln 57 . . B B. . . 0.97 0.37 . . F 0.68 1.91 Ser 58 . . . . . T C 1.82 −0.24 . . F2.22 2.39 Ser 59 . . . . . T C 2.12 −0.60 * . F 2.86 2.22 Gly 60 . . . .T T . 2.48 −0.61 * . F 3.40 1.72 Arg 61 . . . . T T . 1.89 −1.01 * . F3.06 2.51 Asn 62 . . . . . T C 1.08 −0.90 * . F 2.52 1.89 Ser 63 . . B .. T . 0.57 −0.60 * . F 1.98 1.58 Arg 64 . . B . . T . 0.57 −0.34 * . F1.19 0.66 Ala 65 . . B . . T . 0.67 0.04 * * . 0.10 0.55 Leu 66 . . B .. . . 0.56 0.40 * * . −0.10 0.65 Leu 67 . . B . . . . 0.21 0.01 . * .−0.10 0.55 Ser 68 . . B . . T . −0.30 0.44 . * . −0.20 0.54 Tyr 69 . . B. . T . −0.41 0.63 . * . −0.20 0.54 Asp 70 . . . . T T . 0.18 0.34 . . F0.80 1.06 Gly 71 . . . . T T . 1.10 0.06 . * F 0.80 1.36 Leu 72 . . . .. . C 1.06 −0.33 * * F 1.00 1.70 Asn 73 . . B B . . . 1.47 −0.44 * * F0.45 0.76 Gln 74 . . B B . . . 0.86 −0.44 * * F 0.60 1.50 Arg 75 . . B B. . . 0.04 −0.23 * * F 0.60 1.35 Val 76 . A B B . . . 0.39 −0.23 * * .0.30 0.69 Arg 77 . A B B . . . 1.20 −0.63 * * . 0.60 0.67 Val 78 . A B B. . . 1.31 −1.03 . * . 0.60 0.59 Leu 79 . A B B . . . 1.36 −1.03 . * .0.75 1.56 Asp 80 . A B B . . . 0.66 −1.67 . * F 0.90 1.59 Glu 81 . A B .T . . 0.70 −1.17 * . F 1.30 2.16 Arg 82 . A . . T . . −0.30 −1.13 * . F1.30 2.16 Lys 83 . A . . T . . 0.34 −1.13 . . F 1.15 0.91 Ala 84 . A . .T . . 0.49 −0.70 . * . 1.00 0.81 Leu 85 . A B . . . . 0.53 −0.13 * . .0.30 0.22 Ile 86 . . B . . T . 0.64 −0.13 * . . 0.70 0.22 Pro 87 . . B .. T . −0.28 −0.13 * . . 0.70 0.43 Cys 88 . . B . . T . −1.02 0.06 * . .0.10 0.43 Lys 89 . . B . . T . −0.43 0.16 * . . 0.10 0.53 Arg 90 . . B .. . . 0.13 −0.53 * . . 0.80 0.60 Leu 91 . . B B . . . 0.13 −0.20 * . .0.45 1.74 Phe 92 . . B B . . . −0.47 −0.09 * . . 0.30 0.61 Glu 93 . . BB . . . −0.61 0.60 * . . −0.60 0.26 Tyr 94 . . B B . . . −0.90 1.29 * .. −0.60 0.26 Ile 95 . . B B . . . −0.97 1.36 * . . −0.60 0.46 Leu 96 . .B B . . . −0.16 0.57 . . . −0.60 0.54 Leu 97 . . B B . . . 0.20 0.57 . .. −0.60 0.57 Tyr 98 . . . . T T . −0.66 0.24 . . . 0.50 0.81 Lys 99 . .. . T T . −1.01 0.20 . . . 0.50 0.73 Asp 100 . . . . T T . −0.82 0.13 .. . 0.50 0.87 Gly 101 . . B . . T . −0.01 0.23 . * . 0.10 0.48 Val 102 .. B B . . . −0.09 −0.13 . * . 0.30 0.42 Met 103 . . B B . . . 0.16 0.56. * . −0.60 0.18 Phe 104 . . B B . . . 0.11 0.56 . * . −0.60 0.30 Gln105 . . B B . . . −0.48 0.53 * . . −0.30 0.69 Ile 106 . . B B . . .−0.44 0.39 * * . 0.30 0.70 Asp 107 . . B B . . . 0.46 0.26 * * F 0.901.17 Gln 108 . . . . T . . 1.06 −0.53 * * F 2.70 1.36 Ala 109 . . . . T. . 1.09 −0.53 * * F 3.00 3.35 Thr 110 . . . . T . . 0.79 −0.64 * . F2.70 1.07 Lys 111 . . . . T . . 1.72 −0.26 * . F 1.95 0.83 Gln 112 . . .. T . . 1.12 −0.66 * . F 2.10 1.65 Cys 113 . . . . T . . 0.81 −0.54 * *F 1.80 1.13 Ser 114 . . B . . . . 0.59 −0.54 * * F 0.95 0.81 Lys 115 . .B . . . . 0.59 0.14 . . F 0.05 0.39 Met 116 . . B . . . . 0.54 0.23 . .. 0.05 1.04 Thr 117 . . B . . . . 0.33 0.06 . . . 0.05 1.35 Leu 118 . .B . . . . 0.71 0.10 . * F 0.20 1.04 Thr 119 . . B . . . . 1.01 1.01 . *F −0.10 1.11 Gln 120 . . B . . . . 0.76 0.40 * * F 0.20 1.28 Pro 121 . .. . T . . 0.54 0.34 * . F 0.60 2.41 Trp 122 . . . . T . . 0.86 0.34 * .F 0.60 1.37 Asp 123 . . . . . T C 0.78 −0.14 * . F 1.20 1.33 Pro 124 . .B . . T . 0.88 0.14 . . F 0.25 0.60 Leu 125 . . . . T T . 0.88 0.14 . .F 0.93 0.88 Asp 126 . . B . . T . 1.09 −0.37 . . F 1.41 0.92 Ile 127 . .. . . . C 1.08 0.03 . . F 1.09 0.95 Pro 128 . . . . . T C 0.77 −0.01 . *F 2.32 1.55 Gln 129 . . . . T T . 0.28 −0.21 . * F 2.80 1.34 Asn 130 . .. . . T C 1.09 0.57 . * F 1.42 1.65 Ser 131 . . . . . T C 1.09 −0.11 . *F 2.04 1.85 Thr 132 . . B . . . . 1.98 −0.54 . . F 1.66 1.79 Phe 133 . .B . . . . 1.94 −0.54 . . F 1.60 1.92 Glu 134 . . B . . . . 1.64 −0.19. * F 1.24 2.25 Asp 135 . . B . . T . 0.76 −0.19 . * F 1.66 2.09 Gln 136. . B . . T . 0.71 0.01 . * . 1.13 1.69 Tyr 137 . . . . T T . 0.68 −0.34. * . 2.20 0.97 Ser 138 . . . . T T . 1.17 0.09 . * . 1.38 0.57 Ile 139. . . . T . . 1.17 0.51 . . F 0.97 0.51 Gly 140 . . . . . . C 1.17 0.51. . F 0.71 0.56 Gly 141 . . . . . . C 1.17 −0.24 . * F 1.55 0.73 Pro 142. . . . . . C 0.52 −0.23 . . F 1.64 1.80 Gln 143 . . . B . . C 0.51−0.23 . * F 1.60 1.28 Glu 144 . . B B . . . 0.54 −0.17 . * F 1.24 1.86Gln 145 . . B B . . . 0.89 0.04 . * F 0.33 0.89 Ile 146 . . B B . . .1.23 0.01 . * F 0.17 0.89 Thr 147 . . B B . . . 1.16 −0.39 . * . 0.460.89 Val 148 . . B B . . . 0.86 0.53 . * . −0.60 0.54 Gln 149 . . B B .. . 0.86 0.51 . * . −0.11 1.04 Glu 150 . . B B . . . 0.97 −0.17 . * F1.28 1.20 Trp 151 . . . . T T . 1.90 −0.66 . . F 2.72 3.17 Ser 152 . . .. . T C 1.91 −1.30 . . F 2.86 3.66 Asp 153 . . . . T T . 2.18 −1.31 * *F 3.40 2.83 Arg 154 . . . . T T . 2.29 −0.81 * * F 3.06 2.72 Lys 155 . .. . T . . 1.99 −1.73 * * F 2.72 3.98 Ser 156 . . . . . . C 2.03−1.73 * * F 2.38 3.19 Ala 157 . . . . . T C 2.33 −0.97 * . F 2.44 2.55Arg 158 . . . . . T C 2.02 −0.97 * * F 2.30 2.21 Ser 159 . . B . . T .1.62 −0.49 * * F 2.00 2.38 Tyr 160 . . B . . T . 0.69 0.04 * . F 1.202.48 Glu 161 . . B B . . . 0.64 0.23 * * F 0.45 0.89 Thr 162 . . B B . .. 0.34 0.66 * . . −0.20 0.65 Trp 163 . . B B . . . −0.01 0.96 * . .−0.40 0.29 Ile 164 . . B B . . . −0.02 0.96 . . . −0.60 0.27 Gly 165 . .B B . . . −0.63 1.44 . . . −0.60 0.27 Ile 166 . . B B . . . −0.59 1.60 .. . −0.60 0.19 Tyr 167 . . B B . . . −0.28 0.69 . . . −0.60 0.53 Thr 168. . B B . . . −0.66 −0.00 . . . 0.30 0.90 Val 169 . . . B T . . −0.010.14 . . . 0.24 0.69 Lys 170 . . . . T T . 0.12 0.21 * . F 0.93 0.69 Asp171 . . . . T T . 0.16 −0.11 . * . 1.52 0.74 Cys 172 . . B . . T . 0.400.04 . * . 0.66 0.74 Tyr 173 . . B . . T . 0.71 −0.20 * . . 1.40 0.64Pro 174 . . B . . . . 1.26 −0.20 * * . 1.06 0.66 Val 175 . . B B . . .0.51 0.29 * . . 0.27 1.78 Gln 176 . . B B . . . 0.20 0.50 . * F −0.170.99 Glu 177 . . B B . . . −0.02 0.23 . * F −0.01 0.92 Thr 178 . . B B .. . 0.22 0.49 . * F −0.45 0.87 Phe 179 . . B B . . . 0.19 0.24 . * .−0.30 0.81 Thr 180 . . B B . . . 0.74 0.60 . * . −0.60 0.73 Ile 181 . .B B . . . −0.11 0.99 . * . −0.60 0.68 Asn 182 . . B B . . . −1.00 1.14. * . −0.60 0.58 Tyr 183 . . B B . . . −1.50 1.04 . * . −0.60 0.28 Ser184 . . B B . . . −1.10 1.24 . * . −0.60 0.33 Val 185 . . B B . . .−1.10 0.94 * * . −0.60 0.28 Ile 186 . . B B . . . −0.10 1.03 * * . −0.600.25 Leu 187 . . B B . . . −0.80 0.27 * * . −0.30 0.37 Ser 188 . . B B .. . −1.26 0.67 * * . −0.60 0.43 Thr 189 . . B B . . . −0.96 0.81 * * F−0.45 0.54 Arg 190 . . B B . . . −0.99 0.13 . * . −0.15 1.09 Phe 191 . .B B . . . −0.10 0.13 . * . −0.30 0.57 Phe 192 . . B B . . . −0.10 0.14. * . −0.30 0.68 Asp 193 . . B B . . . −0.14 0.34 * * . −0.30 0.29 Ile194 . . B B . . . −0.72 0.77 . * . −0.60 0.33 Gln 195 . . B B . . .−0.79 0.67 * * . −0.60 0.27 Leu 196 . . B B . . . −0.09 −0.11 * * . 0.510.32 Gly 197 . . . . T . . 0.40 −0.11 . * . 1.32 0.76 Ile 198 . . . . T. . 0.10 −0.37 . * . 1.53 0.68 Lys 199 . . . . . . C 0.13 −0.39 * * F1.84 1.10 Asp 200 . . . . . T C −0.57 −0.43 . * F 2.10 0.83 Pro 201 . .B . . T . −0.07 −0.07 . . F 1.84 1.02 Ser 202 . . B . . T . 0.07 −0.27 .. F 1.48 0.74 Val 203 . . B . . T . 0.74 0.16 . . . 0.52 0.68 Phe 204 .. B . . . . 0.40 0.59 . . . −0.19 0.68 Thr 205 . . . . . . C 0.09 0.54 .. F −0.05 0.68 Pro 206 . . . . . T C −0.37 0.64 . . F 0.30 1.32 Pro 207. . . . T T . −0.07 0.57 . . F 0.35 0.82 Ser 208 . . . . T T . 0.19 0.19. . F 0.65 0.98 Thr 209 . . . . T T . 0.30 0.31 . . F 0.65 0.63 Cys 210. A B . . . . 0.61 0.39 . . . −0.30 0.41 Gln 211 . A B . . . . 0.01 0.36. . . −0.30 0.53 Met 212 . A B . . . . 0.22 0.66 . . . −0.60 0.30 Ala213 . A B . . . . 0.57 0.17 . . . −0.30 0.98 Gln 214 A A . . . . . 0.28−0.40 . . . 0.45 1.13 Leu 215 A A . . . . . 0.64 −0.19 . . . 0.76 1.13Glu 216 . A B . . . . 0.64 −0.41 * . . 1.07 1.50 Lys 217 . A B . . . .1.24 −0.91 * . F 1.83 1.50 Met 218 . A . . T . . 1.17 −1.31 * . F 2.543.05 Ser 219 . . . . T T . 0.87 −1.43 * . F 3.10 0.94 Glu 220 . . . . TT . 1.39 −1.04 * . F 2.79 0.63 Asp 221 . . . . T T . 1.00 −0.13 . * F2.18 0.67 Cys 222 . . . . T T . 0.57 −0.31 . * . 1.72 0.64 Ser 223 . . .. T . . 0.78 −0.27 . . . 1.21 0.47 Trp 224 . . . . T . . 0.69 0.16 . . .0.30 0.36

Among highly preferred fragments in this regard are those that compriseregions of Human Ependymin that combine several structural features,such as several of the features set out above.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, the cDNAclone contained in ATCC Deposit No. 209464. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 (e.g., 50) nt of the reference polynucleotide. These areuseful as diagnostic probes and primers as discussed above and in moredetail below.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in FIGS. 1A, 1B, and 1C (SEQ IDNO:1)). Of course, a polynucleotide which hybridizes only to a poly Asequence (such as the 3′ terminal poly(A) tract of the Ependymin cDNAshown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1)), or to a complementarystretch of T (or U) residues, would not be included in a polynucleotideof the invention used to hybridize to a portion of a nucleic acid of theinvention, since such a polynucleotide would hybridize to any nucleicacid molecule containing a poly (A) stretch or the complement thereof(e.g., practically any double-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention whichencode a Ependymin polypeptide may include, but are not limited to thoseencoding the amino acid sequence of the mature polypeptide, by itself;and the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about 37 amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro-protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences.

Also encoded by nucleic acids of the invention are the above proteinsequences together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; anadditional coding sequence which codes for additional amino acids, suchas those which provide additional functionalities.

Thus, the sequence encoding the polypeptide may be fused to a markersequence, such as a sequence encoding a peptide which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described by Gentzand colleagues (Proc. Natl. Acad. Sci. USA 86:821-824 (1989)), forinstance, hexa-histidine provides for convenient purification of thefusion protein. The “HA” tag is another peptide useful for purificationwhich corresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson and coworkers (Cell 37:767(1984)). As discussed below, other such fusion proteins include theEpendymin fused to Fc at the N- or C-terminus.

Variant and Mutant Polynucleotides

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the Ependymin protein. Variants may occur naturally, suchas a natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985)). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the Ependymin protein or portions thereof.Also especially preferred in this regard are conservative substitutions.

Most highly preferred are nucleic acid molecules encoding the matureprotein having the amino acid sequence shown in SEQ ID NO:2 or themature Ependymin amino acid sequence encoded by the deposited cDNAclone.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the Ependymin polypeptide having the complete amino acidsequence in SEQ ID NO:2 (i.e., positions −37 to 187 of SEQ ID NO:2); (b)a nucleotide sequence encoding the Ependymin polypeptide having thecomplete amino acid sequence in SEQ ID NO:2 excepting the N-terminalmethionine (i.e., positions −36 to 187 of SEQ ID NO:2); (c) a nucleotidesequence encoding the predicted mature Ependymin polypeptide having theamino acid sequence at positions 1 to 187 in SEQ ID NO:2; (d) anucleotide sequence encoding the Ependymin polypeptide having thecomplete amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 209464; (e) a nucleotide sequence encoding the Ependyminpolypeptide having the complete amino acid sequence excepting theN-terminal methionine encoded by the cDNA clone contained in ATCCDeposit No. 209464; (f) a nucleotide sequence encoding the matureEpendymin polypeptide having the amino acid sequence encoded by the cDNAclone contained in ATCC Deposit No. 209464; and (g) a nucleotidesequence complementary to any of the nucleotide sequences in (a), (b),(c), (d), (e) or (f), above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical, to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f) or (g), above, or a polynucleotide which hybridizes understringent hybridization conditions to a polynucleotide in (a), (b), (c),(d), (e), (f) or (g), above. This polynucleotide which hybridizes doesnot hybridize under stringent hybridization conditions to apolynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues. An additional nucleic acid embodiment ofthe invention relates to an isolated nucleic acid molecule comprising apolynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a Ependymin polypeptide having an amino acidsequence in (a), (b), (c), (d), (e) or (f), above. A further nucleicacid embodiment of the invention relates to an isolated nucleic acidmolecule comprising a polynucleotide which encodes the amino acidsequence of a Ependymin polypeptide having an amino acid sequence whichcontains at least one conservative amino acid substitution, but not morethan 50 conservative amino acid substitutions, even more preferably, notmore than 40 conservative amino acid substitutions, still morepreferably, not more than 30 conservative amino acid substitutions, andstill even more preferably, not more than 20 conservative amino acidsubstitutions. Of course, in order of ever-increasing preference, it ishighly preferable for a polynucleotide which encodes the amino acidsequence of a Ependymin polypeptide to have an amino acid sequence whichcontains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservativeamino acid substitutions.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofEpendymin polypeptides or peptides by recombinant techniques.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a Ependyminpolypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding theEpendymin polypeptide. In other words, to obtain a polynucleotide havinga nucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in FIGS. 1A, 1B, and 1C, or to the nucleotidessequence of the deposited cDNA clone HDPIE88 can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman to find the best segment of homology between two sequences(Advances in Applied Mathematics 2:482-489 (1981)). When using Bestfitor any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference nucleotide sequence and that gaps in homology ofup to 5% of the total number of nucleotides in the reference sequenceare allowed. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag and colleagues (Comp. App. Biosci. 6:237-245 (1990)). In asequence alignment the query and subject sequences are both DNAsequences. An RNA sequence can be compared by converting U's to T's. Theresult of said global sequence alignment is in percent identity.Preferred parameters used in a FASTDB alignment of DNA sequences tocalculate percent identity are: Matrix=Unitary, k-tuple=4, MismatchPenalty=1, Joining Penalty=30, Randomization Group Length=0, CutoffScore=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or thelength of the subject nucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) or to the nucleic acidsequence of the deposited cDNA, irrespective of whether they encode apolypeptide having Ependymin activity. This is because even where aparticular nucleic acid molecule does not encode a polypeptide havingEpendymin activity, one of skill in the art would still know how to usethe nucleic acid molecule, for instance, as a hybridization probe or apolymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving Ependymin activity include, inter alia, (1) isolating theEpendymin gene or allelic variants thereof in a cDNA library; (2) insitu hybridization (e.g., “FISH”) to metaphase chromosomal spreads toprovide precise chromosomal location of the Ependymin gene, as describedby Verma and colleagues (Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York (1988)); and Northern Blot analysisfor detecting Ependymin mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) or to the nucleic acidsequence of the deposited cDNA which do, in fact, encode a polypeptidehaving Ependymin protein activity. By “a polypeptide having Ependyminactivity” is intended polypeptides exhibiting activity similar, but notnecessarily identical, to an activity of the mature Ependymin protein ofthe invention, as measured in a particular biological assay. Forexample, the Ependymin protein of the present invention binds Ca²⁺ to ahigh degree. Maruyama and colleagues (J. Biochem. 95:511-519 (1984)),Ganss and Hoffman (Eur. J. Biochem. 217:275-280 (1993)), and Schmidt andMakiola (Neuro. Chem. (Life Sci. Adv.) 10:161-171 (1991)) eachdemonstrate a convenient laboratory method to determine and roughlyquantitate the amount of radiolabeled Ca²⁺ which a given polypeptide,such as Ependymin of the present invention, can bind. Briefly, 5-10 μgprotein is used for each analysis. The protein samples are separated byconventional SDS/PAGE using a 13% polyacrylamide gel according tomethods which are well-known to one of ordinary skill in the art.Following SDS/PAGE, the proteins are transferred to a nitrocellulosefilter also according to methods which are well-known to one of ordinaryskill in the art (for example, such a method is provided by Towbin, H.,et al., Proc. Natl. Acad. Sci. USA 76:4350-4354 (1979)). After theprotein is transferred to the nitrocellulose filter, the blot ishybridized with 200 ml ⁴⁵CaCl₂ (300 μCi) in the presence of 5 mM MgCl₂.To reduce non-specific binding of Ca²⁺, the transfer buffer is washedout by three changes (20 minutes each) of 10 mM imidazole/HCl buffer, pH6.8, containing 5 mM MgCl₂ and 60 mM KCl. In such analyses, it isconvenient to use a well-known, strong Ca²⁺-binding protein, such ascalmodulin (Pharmacia) and a well-known, protein which does not bindCa²⁺, such as bovine serum albumin (Sigma) as controls. To quantitatethe amount of Ca²⁺ bound to a specific protein, the region of thenitrocellulose filter which to which the protein is bound is cut out andplaced in a standard scintillation vial. The vial is then filled with 4ml scintillation cocktail (for example, Quickszint 361; Zinsser,Maidenhead, U. K.) and counted in a liquid scintillation counter (forexample, the Wallac 1410, Pharmacia). Using such an analysis, one ofordinary skill in the art may easily ascertain useful qualitative andquantitative information regarding proteins such as Ependymin, ormuteins thereof, of the present invention such as the amount of Ca²⁺that the protein will bind and the apparent molecular mass of theprotein. Ca²⁺-binding activity is a useful indicator of the potentialfor more complex biological activities.

Ependymin protein binds Ca²⁺ in a dose-dependent manner in theabove-described assay. Thus, “a polypeptide having Ependymin proteinactivity” includes polypeptides that also exhibit any of the sameCa²⁺-binding activities in the above-described assays in adose-dependent manner. Although the degree of dose-dependent activityneed not be identical to that of the Ependymin protein, preferably, “apolypeptide having Ependymin protein activity” will exhibitsubstantially similar dose-dependence in a given activity as compared tothe Ependymin protein (i.e., the candidate polypeptide will exhibitgreater activity or not more than about 25-fold less and, preferably,not more than about tenfold less activity relative to the referenceEpendymin protein).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the deposited cDNAor the nucleic acid sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1)will encode a polypeptide “having Ependymin protein activity.” In fact,since degenerate variants of these nucleotide sequences all encode thesame polypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving Ependymin protein activity. This is because the skilled artisanis fully aware of amino acid substitutions that are either less likelyor not likely to significantly effect protein function (e.g., replacingone aliphatic amino acid with a second aliphatic amino acid), as furtherdescribed below.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of Ependyminpolypeptides or fragments thereof by recombinant techniques. The vectormay be, for example, a phage, plasmid, viral or retroviral vector.Retroviral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe transcripts expressed by the constructs will preferably include atranslation initiating codon at the beginning and a termination codon(UAA, UGA or UAG) appropriately positioned at the end of the polypeptideto be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

Vectors preferred for use in bacteria include pHE4-5 (ATCC Accession No.209311; and variations thereof), pQE70, pQE60 and pQE-9 (QIAGEN, Inc.,supra); pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); and ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Among preferred eukaryotic vectors arepWLNEO, pSV2CAT, pOG44, pXT1, and pSG (Stratagene); and pSVK3, pBPV,pMSG and pSVL (Pharmacia). Other suitable vectors will be readilyapparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals (for example, Davis, et al., Basic Methods InMolecular Biology (1986)).

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to stabilize andpurify proteins. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobulin molecules together with another humanprotein or part thereof. In many cases, the Fc part in a fusion proteinis thoroughly advantageous for use in therapy and diagnosis and thusresults, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be ableto delete the Fc part after the fusion protein has been expressed,detected and purified in the advantageous manner described. This is thecase when Fc portion proves to be a hindrance to use in therapy anddiagnosis, for example when the fusion protein is to be used as antigenfor immunizations. In drug discovery, for example, human proteins, suchas hIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5(Bennett, D., et al., J. Molecular Recognition 8:52-58 (1995); Johanson,K., et al., J. Biol. Chem. 270:9459-9471 (1995)).

The Ependymin protein can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification. Polypeptides ofthe present invention include: products purified from natural sources,including bodily fluids, tissues and cells, whether directly isolated orcultured; products of chemical synthetic procedures; and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes. Thus, it is wellknown in the art that the N-terminal methionine encoded by thetranslation initiation codon generally is removed with high efficiencyfrom any protein after translation in all eukaryotic cells. While theN-terminal methionine on most proteins also is efficiently removed inmost prokaryotes, for some proteins this prokaryotic removal process isinefficient, depending on the nature of the amino acid to which theN-terminal methionine is covalently linked.

Polypeptides and Fragments

The invention further provides an isolated Ependymin polypeptide havingthe amino acid sequence encoded by the deposited cDNA, or the amino acidsequence in SEQ ID NO:2, or a peptide or polypeptide comprising aportion of the above polypeptides.

Variant and Mutant Polypeptides

To improve or alter the characteristics of Ependymin polypeptides,protein engineering may be employed. Recombinant DNA technology known tothose skilled in the art can be used to create novel mutant proteins ormuteins including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

N-Terminal and C-Terminal Deletion Mutants

For instance, for many proteins, including the extracellular domain of amembrane associated protein or the mature form(s) of a secreted protein,it is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron and colleagues (J. Biol. Chem.,268:2984-2988 (1993)) reported modified KGF proteins that had heparinbinding activity even if 3, 8, or 27 N-terminal amino acid residues weremissing. In the present case, since the protein of the invention is amember of the ependymin polypeptide family, deletions of N-terminalamino acids up to the cysteine at position 5 of SEQ ID NO:2 may retainsome biological activity such as Ca²⁺-binding or the ability to modulateregeneration. Polypeptides having further N-terminal deletions includingthe Cys-5 residue in SEQ ID NO:2 would not be expected to retain suchbiological activities because this residue is conserved in each of theependymin-related polypeptides shown in FIGS. 3A and 3B and it is likelythat a cysteine in such a position may be required for forming adisulfide bridge to provide structural stability which is needed forreceptor binding and signal transduction.

However, even if deletion of one or more amino acids from the N-terminusof a protein results in modification or loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or mature of the proteingenerally will be retained when less than the majority of the residuesof the complete or mature protein are removed from the N-terminus.Whether a particular polypeptide lacking N-terminal residues of acomplete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence of the Ependymin shown in SEQ ID NO:2, up to the cysteineresidue at position number 5, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues n¹-187 of SEQ ID NO:2,where n¹ is an integer in the range of −37 to 4, and 5 is the positionof the first residue from the N-terminus of the complete Ependyminpolypeptide (shown in SEQ ID NO:2) believed to be required for theCa²⁺-binding or the regeneration modulatory activities of the Ependyminprotein.

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues of −37-187,−36-187, −35-187, −34-187, −187, −32-187, −31-187, −30-187, −29-187,−28-187, −27-187, −26 -187, −25-187, −24-187, −23-187, −22-187, −21-187,−20-187, −19-187, −18-187, −17-187, −16-187, −15-187, −14-187, −13-187,−12-187, −11-187, −10-187, −9-187, −8-187, −7-187, −6-187, −5-187,−4-187, −3-187, −2-187, −1-187, −1-187, 2-187, 3-187, 4-187, and 5-187of SEQ ID NO:2. Polynucleotides encoding these polypeptides also areprovided.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. For instance, Interferon gamma shows up to ten timeshigher activities by deleting 8-10 amino acid residues from the carboxyterminus of the protein (Dobeli, et al., J. Biotechnology 7:199-216(1988)). In the present case, since the protein of the invention is amember of the ependymin polypeptide family, deletions of C-terminalamino acids up to the cysteine at position 173 of SEQ ID NO:2 may retainsome biological activity such as Ca²⁺-binding or the ability to modulateregeneration. Polypeptides having further C-terminal deletions includingthe cysteine residue at position 173 of SEQ ID NO:2 would not beexpected to retain such biological activities because this residue isconserved in each of the ependymin-related polypeptides shown in FIGS.3A and 3B and it is likely that a cysteine in such a position may berequired for forming a disulfide bridge to provide structural stabilitywhich is needed for receptor binding and signal transduction.

However, even if deletion of one or more amino acids from the C-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or mature of the proteingenerally will be retained when less than the majority of the residuesof the complete or mature protein are removed from the C-terminus.Whether a particular polypeptide lacking C-terminal residues of acomplete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

Accordingly, the present invention further provides polypeptides havingone or more residues from the carboxy terminus of the amino acidsequence of the Ependymin shown in SEQ ID NO:2, up to the cysteineresidue at position 173 of SEQ ID NO:2, and polynucleotides encodingsuch polypeptides. In particular, the present invention providespolypeptides having the amino acid sequence of residues −37-m¹ of theamino acid sequence in SEQ ID NO:2, where m¹ is any integer in the rangeof 173 to 187, and residue 173 is the position of the first residue fromthe C-terminus of the complete Ependymin polypeptide (shown in SEQ IDNO:2) believed to be required for the Ca²⁺-binding or the regenerationmodulatory abilities of the Ependymin protein.

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues −37-173,−37-174, −37-175, −37-176, −37-177, −37-178, −37-179, −37-180, −37-181,−37-182, −37-183, −37-184, −37-184, −37-185, −37-186, and −37-187 of SEQID NO:2. Polynucleotides encoding these polypeptides also are provided.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues n¹-m¹ of SEQ ID NO:2, where n¹and m¹ are integers as described above.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete Ependymin amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 209464, wherethis portion excludes from 1 to about 42 amino acids from the aminoterminus of the complete amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 209464, or from 1 to about 15 amino acidsfrom the carboxy terminus, or any combination of the above aminoterminal and carboxy terminal deletions, of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209464.Polynucleotides encoding all of the above deletion mutant polypeptideforms also are provided.

As mentioned above, even if deletion of one or more amino acids from theN-terminus of a protein results in modification of loss of one or morebiological functions of the protein, other biological activities maystill be retained. Thus, the ability of the shortened Human Ependyminmutein to induce and/or bind to antibodies which recognize the completeor mature of the protein generally will be retained when less than themajority of the residues of the complete or mature protein are removedfrom the N-terminus. Whether a particular polypeptide lacking N-terminalresidues of a complete protein retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a Human Ependymin mutein witha large number of deleted N-terminal amino acid residues may retain somebiological or immungenic activities. In fact, peptides composed of asfew as six Human Ependymin amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the HumanEpendymin amino acid sequence shown in SEQ ID NO:2, up to the serineacid residue at position number 219 and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues n²-224 of FIGS. 1A, 1B,and 1C (SEQ ID NO:2), where n² is an integer in the range of 2 to 219,and 220 is the position of the first residue from the N-terminus of thecomplete Human Ependymin polypeptide believed to be required for atleast immunogenic activity of the Human Ependymin protein.

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues of P-2 to W-224; G-3 to W-224; R-4 to W-224; A-5 toW-224; P-6 to W-224; L-7 to W-224; R-8 to W-224; T-9 to W-224; V-10 toW-224; P-11 to W-224; G-12 to W-224; A-13 to W-224; L-14 to W-224; G-15to W-224; A-16 to W-224; W-17 to W-224; L-18 to W-224; L-19 to W-224;G-20 to W-224; G-21 to W-224; L-22 to W-224; W-23 to W-224; A-24 toW-224; W-25 to W-224; T-26 to W-224; L-27 to W-224; C-28 to W-224; G-29W-224; L-30 to W-224; C-31 to W-224; S-32 to W-224; L-33 to W-224; G-34to W-224; A-35 to W-224; V-36 to W-224; G-37 to W-224; A-38 to W-224;P-39 to W-224; R-40 to W-224; P-41 to W-224; C-42 to W-224; Q-43 toW-224; A-44 to W-224; P-45 to W-224; Q-46 to W-224; Q-47 to W-224; W-48to W-224; E-49 to W-224; G-50 to W-224; R-51 to W-224; Q-52 to W-224;V-53 to W-224; M-54 to W-224; Y-55 to W-224; Q-56 to W-224; Q-57 toW-224; S-58 to W-224; S-59 to W-224; G-60 to W-224; R-61 to W-224; N-62to W-224; S-63 to W-224; R-64 to W-224; A-65 to W-224; L-66 to W-224;L-67 to W-224; S-68 to W-224; Y-69 to W-224; D-70 to W-224; G-71 toW-224; L-72 to W-224; N-73 to W-224; Q-74 to W-224; R-75 to W-224; V-76to W-224; R-77 to W-224; V-78 to W-224; L-79 to W-224; D-80 to W-224;E-81 to W-224; R-82 to W-224; K-83 to W-224; A-84 to W-224; L-85 toW-224; I-86 to W-224; P-87 to W-224; C-88 to W-224; K-89 to W-224; R-90to W-224; L-91 to W-224; F-92 to W-224; E-93 to W-224; Y-94 to W-224;1-95 to W-224; L-96 to W-224; L-97 to W-224; Y-98 to W-224; K-99 toW-224; D-100 to W-224; G-101 to W-224; V-102 to W-224; M-103 to W-224;F-104 to W-224; Q-105 to W-224; I-106 to W-224; D-107 to W-224; Q-108 toW-224; A-109 to W-224; T-110 to W-224; K-111 to W-224; Q-112 to W-224;C-113 to W-224; S-114 to W-224; K-115 to W-224; M-116 to W-224; T-117 toW-224; L-118 to W-224; T-119 to W-224; Q-120 to W-224; P-121 to W-224;W-122 to W-224; D-123 to W-224; P-124 to W-224; L-125 to W-224; D-126 toW-224; I-127 to W-224; P-128 to W-224; Q-129 to W-224; N-130 to W-224;S-131 to W-224; T-132 to W-224; F-133 to W-224; E-134 to W-224; D-135 toW-224; Q-136 to W-224; Y-137 to W-224; S-138 to W-224; I-139 to W-224;G-140 to W-224; G-141 to W-224; P-142 to W-224; Q-143 to W-224; E-144 toW-224; Q-145 to W-224; I-146 to W-224; T-147 to W-224; V-148 to W-224;Q-149 to W-224; E-150 to W-224; W-151 to W-224; S-152 to W-224; D-153 toW-224; R-154 to W-224; K-155 to W-224; S-156 to W-224; A-157 to W-224;R-158 to W-224; S-159 to W-224; Y-160 to W-224; E-161 to W-224; T-162 toW-224; W-163 to W-224; I-164 to W-224; G-165 to W-224; I-166 to W-224;Y-167 to W-224; T-168 to W-224; V-169 to W-224; K-170 to W-224; D-171 toW-224; C-172 to W-224; Y-173 to W-224; P-174 to W-224; V-175 to W-224;Q-176 to W-224; E-177 to W-224; T-178 to W-224; F-179 to W-224; T-180 toW-224; I-181 to W-224; N-182 to W-224; Y-183 to W-224; S-184 to W-224;V-185 to W-224; I-186 to W-224; L-187 to W-224; S-188 to W-224; T-189 toW-224; R-190 to W-224; F-191 to W-224; F-192 to W-224; D-193 to W-224;I-194 to W-224; Q-195 to W-224; L-196 to W-224; G-197 to W-224; I-198 toW-224; K-199 to W-224; D-200 to W-224; P-201 to W-224; S-202 to W-224;V-203 to W-224; F-204 to W-224; T-205 to W-224; P-206 to W-224; P-207 toW-224; S-208 to W-224; T-209 to W-224; C-210 to W-224; Q-211 to W-224;M-212 to W-224; A-213 to W-224; Q-214 to W-224; L-215 to W-224; E-216 toW-224; K-217 to W-224; M-218 to W-224; and S-219 to W-224 of the HumanEpendymin amino acid sequence shown in FIGS. 1A, 1B, and 1C (which isidentical to the sequence shown as SEQ ID NO:2, with the exception thatthe amino acid residues in FIGS. 1A, 1B, and 1C are numberedconsecutively from 1 through 224 from the N-terminus to the C-terminus,while the amino acid residues in SEQ ID NO:2 are numbered consecutivelyfrom −37 through 187 to reflect the position of the predicted signalpeptide). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other biological activitiesmay still be retained. Thus, the ability of the shortened HumanEpendymin mutein to induce and/or bind to antibodies which recognize thecomplete or mature of the protein generally will be retained when lessthan the majority of the residues of the complete or mature protein areremoved from the C-terminus. Whether a particular polypeptide lackingC-terminal residues of a complete protein retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a HumanEpendymin mutein with a large number of deleted C-terminal amino acidresidues may retain some biological or immungenic activities. In fact,peptides composed of as few as six Human Ependymin amino acid residuesmay often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the Human Ependymin shown in SEQ ID NO:2, up to the prolineresidue at position number 6, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m² of SEQ ID NO:2,where m² is an integer in the range of 6 to 224, and 6 is the positionof the first residue from the C-terminus of the complete Human Ependyminpolypeptide believed to be required for at least immunogenic activity ofthe Human Ependymin protein.

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues M-1 to S-223; M-1 to C-222; M-1 to D-221; M-1 toE-220; M-1 to S-219; M-1 to M-218; M-1 to K-217; M-1 to E-216; M-1 toL-215; M-1 to Q-214; M-1 to A-213; M-1 to M-212; M-1 to Q-211; M-1 toC-210; M-1 to T-209; M-1 to S-208; M-1 to P-207; M-1 to P-206; M-1 toT-205; M-1 to F-204; M-1 to V-203; M-1 to S-202; M-1 to P-201; M-1 toD-200; M-1 to K-199; M-1 to 1-198; M-1 to G-197; M-1 to L-196; M-1 toQ-195; M-1 to I-194; M-1 to D-193; M-1 to F-192; M-1 to F-191; M-1 toR-190; M-1 to T-189; M-1 to S-188; M-1 to L-187; M-1 to 1-186; M-1 toV-185; M-1 to S-184; M-1 to Y-183; M-1 to N-182; M-1 to I-181; M-1 toT-180; M-1 to F-179; M-1 to T-178; M-1 to E-177; M-1 to Q-176; M-1 toV-175; M-1 to P-174; M-1 to Y-173; M-1 to C-172; M-1 to D-171; M-1 toK-170; M-1 to V-169; M-1 to T-168; M-1 to Y-167; M-1 to I-166; M-1 toG-165; M-1 to I-164; M-1 to W-163; M-1 to T-162; M-1 to E-161; M-1 toY-160; M-1 to S-159; M-1 to R-158; M-1 to A-157; M-1 to S-156; M-1 toK-155; M-1 to R-154; M-1 to D-153; M-1 to S-152; M-1 to W-151; M-1 toE-150; M-1 to Q-149; M-1 to V-148; M-1 to T-147; M-1 to I-146; M-1 toQ-145; M-1 to E-144; M-1 to Q-143; M-1 to P-142; M-1 to G-141; M-1 toG-140; M-1 to I-139; M-1 to S-138; M-1 to Y-137; M-1 to Q-136; M-1 toD-135; M-1 to E-134; M-1 to F-133; M-1 to T-132; M-1 to S-131; M-1 toN-130; M-1 to Q-129; M-1 to P-128; M-1 to I-127; M-1 to D-126; M-1 toL-125; M-1 to P-124; M-1 to D-123; M-1 to W-122; M-1 to P-121; M-1 toQ-120; M-1 to T-119; M-1 to L-118; M-1 to T-117; M-1 to M-116; M-1 toK-115; M-1 to S-114; M-1 to C-113; M-1 to Q-112; M-1 to K-111; M-1 toT-110; M-1 to A-109; M-1 to Q-108; M-1 to D-107; M-1 to I-106; M-1 toQ-105; M-1 to F-104; M-1 to M-103; M-1 to V-102; M-1 to G-101; M-1 toD-100; M-1 to K-99; M-1 to Y-98; M-1 to L-97; M-1 to L-96; M-1 to 1-95;M-1 to Y-94; M-1 to E-93; M-1 to F-92; M-1 to L-91; M-1 to R-90; M-1 toK-89; M-1 to C-88; M-1 to P-87; M-1 to 1-86; M-1 to L-85; M-1 to A-84;M-1 to K-83; M-1 to R-82; M-1 to E-81; M-1 to D-80; M-1 to L-79; M-1 toV-78; M-1 to R-77; M-1 to V-76; M-1 to R-75; M-1 to Q-74; M-1 to N-73;M-1 to L-72; M-1 to G-71; M-1 to D-70; M-1 to Y-69; M-1 to S-68; M-1 toL-67; M-1 to L-66; M-1 to A-65; M-1 to R-64; M-1 to S-63; M-1 to N-62;M-1 to R-61; M-1 to G-60; M-1 to S-59; M-1 to S-58; M-1 to Q-57; M-1 toQ-56; M-1 to Y-55; M-1 to M-54; M-1 to V-53; M-1 to Q-52; M-1 to R-51;M-1 to G-50; M-1 to E-49; M-1 to W-48; M-1 to Q-47; M-1 to Q-46; M-1 toP-45; M-1 to A-44; M-1 to Q-43; M-1 to C-42; M-1 to P-41; M-1 to R-40;M-1 to P-39; M-1 to A-38; M-1 to G-37; M-1 to V-36; M-1 to A-35; M-1 toG-34; M-1 to L-33; M-1 to S-32; M-1 to C-31; M-1 to L-30; M-1 to G-29;M-1 to C-28; M-1 to L-27; M-1 to T-26; M-1 to W-25; M-1 to A-24; M-1 toW-23; M-1 to L-22; M-1 to G-21; M-1 to G-20; M-1 to L-19; M-1 to L-18;M-1 to W-17; M-1 to A-16; M-1 to G-15; M-1 to L-14; M-1 to A-13; M-1 toG-12; M-1 to P-11; M-1 to V-10; M-1 to T-9; M-1 to R-8; M-1 to L-7; M-1to P-6 of the sequence of the Human Ependymin sequence shown in FIGS.1A, 1B, and 1C (which is identical to the sequence shown as SEQ ID NO:2,with the exception that the amino acid residues in FIGS. 1A, 1B, and 1Care numbered consecutively from 1 through 224 from the N-terminus to theC-terminus, while the amino acid residues in SEQ ID NO:2 are numberedconsecutively from −37 through 187 to reflect the position of thepredicted signal peptide). Polynucleotides encoding these polypeptidesalso are provided.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini of a HumanEpendymin polypeptide, which may be described generally as havingresidues n²-m² of FIGS. 1A, 1B, and 1C (SEQ ID NO:2), where n² and m²are integers as described above.

Other Mutants

In addition to terminal deletion forms of the protein discussed above,it also will be recognized by one of ordinary skill in the art that someamino acid sequences of the Ependymin polypeptide can be varied withoutsignificant effect of the structure or function of the protein. If suchdifferences in sequence are contemplated, it should be remembered thatthere will be critical areas on the protein which determine activity.

Thus, the invention further includes variations of the Ependyminpolypeptide which show substantial Ependymin polypeptide activity orwhich include regions of Ependymin protein such as the protein portionsdiscussed below. Such mutants include deletions, insertions, inversions,repeats, and type substitutions selected according to general rulesknown in the art so as have little effect on activity. For example,guidance concerning how to make phenotypically silent amino acidsubstitutions is provided wherein the authors indicate that there aretwo main approaches for studying the tolerance of an amino acid sequenceto change (Bowie, J. U., et al., Science 247:1306-1310 (1990)). Thefirst method relies on the process of evolution, in which mutations areeither accepted or rejected by natural selection. The second approachuses genetic engineering to introduce amino acid changes at specificpositions of a cloned gene and selections or screens to identifysequences that maintain functionality.

As the authors state, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which amino acid changes are likely to be permissive at acertain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described by Bowie and coworkers (supra) and thereferences cited therein. Typically seen as conservative substitutionsare the replacements, one for another, among the aliphatic amino acidsAla, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Thus, the fragment, derivative or analog of the polypeptide of SEQ IDNO:2, or that encoded by the deposited cDNA, may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the above form of the polypeptide, such as an IgG Fc fusionregion peptide or leader or secretory sequence or a sequence which isemployed for purification of the above form of the polypeptide or aproprotein sequence. Such fragments, derivatives and analogs are deemedto be within the scope of those skilled in the art from the teachingsherein.

Thus, the Ependymin of the present invention may include one or moreamino acid substitutions, deletions or additions, either from naturalmutations or human manipulation. As indicated, changes are preferably ofa minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein (seeTable II).

TABLE II Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Amino acids in the Ependymin protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro proliferative activity.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard, et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins, et al., Diabetes 36:838-845 (1987); Cleland, et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993)).

Replacement of amino acids can also change the selectivity of thebinding of a ligand to cell surface receptors (for example, Ostade, etal., Nature 361:266-268 (1993)) describes certain mutations resulting inselective binding of TNF-α to only one of the two known types of TNFreceptors. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith, et al., J. Mol.Biol. 224:899-904 (1992); de Vos, et al. Science 255:306-312 (1992)).

As described above, ependymin function relies, in part, onasparagine-linked glycosylation. Although the relative locations of thetwo potential N-linked glycosylation sites of Ependymin of the presentinvention are not conserved with respect to the other known ependymins,it is likely that disruption of the either or both of the Ependyminglycosylation sites will, at least, modulate Ependymin activity. As aresult, mutation of the asparagine amino acid residues at positions 93and 145 of SEQ ID NO:2 or the threonine or serine amino acid residues atpositions 95 and 147, respectively, will, at least modulate thebiological activity of Ependymin of the present invention.

In addition, as depicted in FIGS. 2 and 3, five cysteine amino acidresidues are conserved between Ependymin of the present invention andfive piscine ependymin homologs. Since cysteine amino acid residuesoften function in a structural capacity with respect to the tertiarystructure of a polypeptide (or the quaternary structure of the samepolypeptide in cases where polymerization is expected), it is expectedthat mutation of any or all of the four conserved cysteine amino acidresidues will result in, at least, modulation of the biological activityof Ependymin of the present invention. As shown in FIGS. 3A and 3B, theconserved cysteine residues are located at positions 5, 51, 76, 135, and173 of SEQ ID NO:2.

Similarly, several additional amino acids are highly conserved betweenEpendymin of the present invention and the five piscine ependyminspresented in FIGS. 3A and 3B. These are Proline-8, Tyrosine-32, AsparticAcid-33, Glycine-64, Isoleucine-69, Aspartic Acid-70, Lysine-78,Leucine-81, Proline-91, Glycine-103, Tryptophan-114, Threonine-131,Phenylalanine-167, Proline-170, and Glutamic Acid-179. It is expectedthat mutation of one, several, or all of these amino acid residues willmodulate the biological activity of Ependymin of the present invention.

Mutations which may also modulate, and are less likely to completelyeliminate, the biological activity of Ependymin of the present inventionmay also be made by changing one or more non-conserved amino acidresidues throughout the Ependymin polypeptide. Also forming part of thepresent invention are isolated polynucleotides comprising nucleic acidsequences which encode the each of the above Ependymin mutants.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of the Ependymin polypeptide can besubstantially purified by the one-step method described by Smith andJohnson (Gene 67:31-40 (1988)). Polypeptides of the invention also canbe purified from natural or recombinant sources using anti-Ependyminantibodies of the invention in methods which are well known in the artof protein purification.

The invention further provides an isolated Ependymin polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of the full-length Ependymin polypeptidehaving the complete amino acid sequence shown in SEQ ID NO:2 (i.e.,positions −37 to 187 of SEQ ID NO:2); (b) the amino acid sequence of thefull-length Ependymin polypeptide having the complete amino acidsequence shown in SEQ ID NO:2 excepting the N-terminal methionine (i.e.,positions -36 to 187 of SEQ ID NO:2); (c) the amino acid sequence of thepredicted mature Ependymin polypeptide having the amino acid sequence atpositions 1 to 187 in SEQ ID NO:2; (d) the complete amino acid sequenceencoded by the cDNA clone contained in the ATCC Deposit No. 209464; (e)the complete amino acid sequence excepting the N-terminal methionineencoded by the cDNA clone contained in the ATCC Deposit No. 209464; and(f) the complete amino acid sequence of the predicted mature Ependyminpolypeptide encoded by the cDNA clone contained in the ATCC Deposit No.209464. The polypeptides of the present invention also includepolypeptides having an amino acid sequence at least 80% identical, morepreferably at least 90% identical, and still more preferably 95%, 96%,97%, 98% or 99% identical to those described in (a), (b), (c), (d), (e)or (f), above, as well as polypeptides having an amino acid sequencewith at least 90% similarity, and more preferably at least 95%similarity, to those above.

Further polypeptides of the present invention include polypeptides whichhave at least 90% similarity, more preferably at least 95% similarity,and still more preferably at least 96%, 97%, 98% or 99% similarity tothose described above. The polypeptides of the invention also comprisethose which are at least 80% identical, more preferably at least 90% or95% identical, still more preferably at least 96%, 97%, 98% or 99%identical to the polypeptide encoded by the deposited cDNA or to thepolypeptide of SEQ ID NO:2, and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

A further embodiment of the invention relates to a peptide orpolypeptide which comprises the amino acid sequence of a Ependyminpolypeptide having an amino acid sequence which contains at least oneconservative amino acid substitution, but not more than 50 conservativeamino acid substitutions, even more preferably, not more than 40conservative amino acid substitutions, still more preferably, not morethan 30 conservative amino acid substitutions, and still even morepreferably, not more than 20 conservative amino acid substitutions. Ofcourse, in order of ever-increasing preference, it is highly preferablefor a peptide or polypeptide to have an amino acid sequence whichcomprises the amino acid sequence of a Ependymin polypeptide, whichcontains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1conservative amino acid substitutions.

By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) tofind the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a Ependyminpolypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the Ependymin polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:2), the aminoacid sequence encoded by deposited cDNA clone HDPIE88, or fragmentsthereof, can be determined conventionally using known computer programssuch the Bestfit program (Wisconsin Sequence Analysis Package, Version 8for Unix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the reference aminoacid sequence and that gaps in homology of up to 5% of the total numberof amino acid residues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. According to this embodiment, if the subject sequence isshorter than the query sequence due to N- or C-terminal deletions, notbecause of internal deletions, a manual correction is made to theresults to take into consideration the fact that the FASTDB program doesnot account for N- and C-terminal truncations of the subject sequencewhen calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal of the subject sequence,which are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. A determination ofwhether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thisembodiment. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence. For example, a 90 aminoacid residue subject sequence is aligned with a 100 residue querysequence to determine percent identity. The deletion occurs at theN-terminus of the subject sequence and therefore, the FASTDB alignmentdoes not show a matching/alignment of the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal deletionsso there are no residues at the N- or C-termini of the subject sequencewhich are not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected for. Noother manual corrections are made for the purposes of this embodiment.

The polypeptide of the present invention could be used as a molecularweight marker on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art.

As described in detail below, the polypeptides of the present inventioncan also be used to raise polyclonal and monoclonal antibodies, whichare useful in assays for detecting Ependymin protein expression asdescribed below or as agonists and antagonists capable of enhancing orinhibiting Ependymin protein function. Further, such polypeptides can beused in the yeast two-hybrid system to “capture” Ependymin proteinbinding proteins which are also candidate agonists and antagonistsaccording to the present invention. The yeast two hybrid system isdescribed by Fields and Song (Nature 340:245-246 (1989)).

Epitope-Bearing Portions

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. On the other hand, a region of aprotein molecule to which an antibody can bind is defined as an“antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes (see, forinstance, Geysen, et al., Proc. Natl. Acad. Sci. USA 81:3998-4002(1983)).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein (see, for instance, Sutcliffe, J. G., et al., Science219:660-666 (1983)). Peptides capable of eliciting protein-reactive seraare frequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are confinedneither to immunodominant regions of intact proteins (i.e., immunogenicepitopes) nor to the amino or carboxyl terminals. Antigenicepitope-bearing peptides and polypeptides of the invention are thereforeuseful to raise antibodies, including monoclonal antibodies, that bindspecifically to a polypeptide of the invention (see, for instance,Wilson, et al., Cell 37:767-778 (1984)).

Antigenic epitope-bearing peptides and polypeptides of the inventionpreferably contain a sequence of at least seven, more preferably atleast nine and most preferably between about 15 to about 30 amino acidscontained within the amino acid sequence of a polypeptide of theinvention. Non-limiting examples of antigenic polypeptides or peptidesthat can be used to generate Ependymin-specific antibodies include: apolypeptide comprising amino acid residues from about Ala-1 to aboutGln-9 in SEQ ID NO:2; a polypeptide comprising amino acid residues fromabout Pro-8 to about Val-16 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about Gln-19 to about Arg-27 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about Ile-69 to aboutSer-77 in SEQ ID NO:2; a polypeptide comprising amino acid residues fromabout Asp-86 to about Glu-107 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about Glu-113 to about Tyr-123 in SEQ ID NO:2;a polypeptide comprising amino acid residues from about Thr-131 to aboutGln-139 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about Leu-159 to about Phe-167 in SEQ ID NO:2; and a polypeptidecomprising amino acid residues from about Leu-178 to about Ser-186 inSEQ ID NO:2. These polypeptide fragments have been determined to bearantigenic epitopes of the Ependymin protein by the analysis of theJameson-Wolf antigenic index, as shown in FIG. 4 and Table I, above.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means (see, for example, Houghten, R. A.,et al., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985); and U.S. Pat.No. 4,631,211 to Houghten, et al. (1986)).

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art (see, forinstance, Sutcliffe, et al., supra; Wilson, et al., supra; Chow, M., etal., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J., et al.,J. Gen. Virol. 66:2347-2354 (1985)). Immunogenic epitope-bearingpeptides of the invention, i.e., those parts of a protein that elicit anantibody response when the whole protein is the immunogen, areidentified according to methods known in the art (see, for instance,Geysen, et al., supra). Further still, U.S. Pat. No. 5,194,392, issuedto Geysen, describes a general method of detecting or determining thesequence of monomers (amino acids or other compounds) which is atopological equivalent of the epitope (i.e., a “mimotope”) which iscomplementary to a particular paratope (antigen binding site) of anantibody of interest. More generally, U.S. Pat. No. 4,433,092, issued toGeysen, describes a method of detecting or determining a sequence ofmonomers which is a topographical equivalent of a ligand which iscomplementary to the ligand binding site of a particular receptor ofinterest. Similarly, U.S. Pat. No. 5,480,971, issued to Houghten andcolleagues, on Peralkylated Oligopeptide Mixtures discloses linearC1-C7-alkyl peralkylated oligopeptides and sets and libraries of suchpeptides, as well as methods for using such oligopeptide sets andlibraries for determining the sequence of a peralkylated oligopeptidethat preferentially binds to an acceptor molecule of interest. Thus,non-peptide analogs of the epitope-bearing peptides of the inventionalso can be made routinely by these methods.

Fusion Proteins

As one of skill in the art will appreciate, Ependymin polypeptides ofthe present invention and the epitope-bearing fragments thereofdescribed above can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EP A 394,827; Traunecker, et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric Ependymin protein orprotein fragment alone (Fountoulakis, et al., J. Biochem. 270:3958-3964(1995)).

Antibodies

Ependymin protein-specific antibodies for use in the present inventioncan be raised against the intact Ependymin protein or an antigenicpolypeptide fragment thereof, which may be presented together with acarrier protein, such as an albumin, to an animal system (such as rabbitor mouse) or, if it is long enough (at least about 25 amino acids),without a carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to Ependymin protein. Fab and F(ab′)2 fragmentslack the Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding of an intactantibody (Wahl, et al., J. Nucl. Med. 24:316-325 (1983)). Thus, thesefragments are preferred.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the Ependymin proteinor an antigenic fragment thereof can be administered to an animal inorder to induce the production of sera containing polyclonal antibodies.In a preferred method, a preparation of Ependymin protein is preparedand purified to render it substantially free of natural contaminants.Such a preparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or Ependymin protein binding fragmentsthereof). Such monoclonal antibodies can be prepared using hybridomatechnology (Kohler, et al., Nature 256:495 (1975); Kohler, et al., Eur.J. Immunol. 6:511 (1976); Kohler, et al., Eur. J. Immunol. 6:292 (1976);Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas,Elsevier, N.Y., (1981) pp. 563-681)). In general, such proceduresinvolve immunizing an animal (preferably a mouse) with a Ependyminprotein antigen or, more preferably, with a Ependymin protein-expressingcell. Suitable cells can be recognized by their capacity to bindanti-Ependymin protein antibody. Such cells may be cultured in anysuitable tissue culture medium; however, it is preferable to culturecells in Earle's modified Eagle's medium supplemented with 10% fetalbovine serum (inactivated at about 56° C.), and supplemented with about10 μg/l of nonessential amino acids, about 1,000 U/ml of penicillin, andabout 100 μg/ml of streptomycin. The splenocytes of such mice areextracted and fused with a suitable myeloma cell line. Any suitablemyeloma cell line may be employed in accordance with the presentinvention; however, it is preferable to employ the parent myeloma cellline (SP2O), available from the American Type Culture Collection,Manassas, Va. After fusion, the resulting hybridoma cells areselectively maintained in HAT medium, and then cloned by limitingdilution as described by Wands and colleagues (Gastroenterology80:225-232 (1981)). The hybridoma cells obtained through such aselection are then assayed to identify clones which secrete antibodiescapable of binding the Ependymin protein antigen.

Alternatively, additional antibodies capable of binding to the Ependyminprotein antigen may be produced in a two-step procedure through the useof anti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and that, therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, Ependymin protein-specific antibodies are used toimmunize an animal, preferably a mouse. The splenocytes of such ananimal are then used to produce hybridoma cells, and the hybridoma cellsare screened to identify clones which produce an antibody whose abilityto bind to the Ependymin protein-specific antibody can be blocked by theEpendymin protein antigen. Such antibodies comprise anti-idiotypicantibodies to the Ependymin protein-specific antibody and can be used toimmunize an animal to induce formation of further Ependyminprotein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, Ependyminprotein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

For in vivo use of anti-Ependymin in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art (Morrison, Science 229:1202(1985); Oi, et al., BioTechniques 4:214 (1986); Cabilly, et al., U.S.Pat. No. 4,816,567; Taniguchi, et al., EP 171496; Morrison, et al., EP173494; Neuberger, et al., WO 8601533; Robinson, et al., WO 8702671;Boulianne, et al., Nature 312:643 (1984); Neuberger, et al., Nature314:268 (1985).

Nervous System-Related Disorders

Diagnosis

The present inventors have discovered that Ependymin is expressed inprimary dendritic cells, the KMH2 cell line, placenta, fetal and adultliver, spinal cord, osteoclastoma, cerebellum, synovial fibroblasts, 12week old early stage human embryo, adrenal gland tumor, whole brain,Hodgkin's Lymphoma tissue, macrophages, HEL cell line, andchondrosarcoma. For a number of nervous system-related disorders,substantially altered (increased or decreased) levels of Ependymin geneexpression can be detected in nervous system tissue or other cells orbodily fluids (e.g., sera, plasma, urine, synovial fluid or spinalfluid) taken from an individual having such a disorder, relative to a“standard” Ependymin gene expression level, that is, the Ependyminexpression level in nervous system tissues or bodily fluids from anindividual not having the nervous system disorder. Thus, the inventionprovides a diagnostic method useful during diagnosis of a nervous systemdisorder, which involves measuring the expression level of the geneencoding the Ependymin protein in nervous system tissue or other cellsor body fluid from an individual and comparing the measured geneexpression level with a standard Ependymin gene expression level,whereby an increase or decrease in the gene expression level compared tothe standard is indicative of an nervous system disorder.

In particular, it is believed that certain tissues in mammals withcancer of the nervous express significantly reduced levels of theEpendymin protein and mRNA encoding the Ependymin protein when comparedto a corresponding “standard” level. Further, it is believed thatenhanced levels of the Ependymin protein can be detected in certain bodyfluids (e.g., sera, plasma, urine, and spinal fluid) from mammals withsuch a cancer when compared to sera from mammals of the same species nothaving the cancer.

Thus, the invention provides a diagnostic method useful during diagnosisof a nervous system disorder, including cancers of this system, whichinvolves measuring the expression level of the gene encoding theEpendymin protein in nervous system tissue or other cells or body fluidfrom an individual and comparing the measured gene expression level witha standard Ependymin gene expression level, whereby an increase ordecrease in the gene expression level compared to the standard isindicative of a nervous system disorder.

Where a diagnosis of a disorder in the nervous system, includingdiagnosis of a tumor, has already been made according to conventionalmethods, the present invention is useful as a prognostic indicator,whereby patients exhibiting depressed Ependymin gene expression willexperience a worse clinical outcome relative to patients expressing thegene at a level nearer the standard level.

By “assaying the expression level of the gene encoding the Ependyminprotein” is intended qualitatively or quantitatively measuring orestimating the level of the Ependymin protein or the level of the mRNAencoding the Ependymin protein in a first biological sample eitherdirectly (e.g., by determining or estimating absolute protein level ormRNA level) or relatively (e.g., by comparing to the Ependymin proteinlevel or mRNA level in a second biological sample). Preferably, theEpendymin protein level or mRNA level in the first biological sample ismeasured or estimated and compared to a standard Ependymin protein levelor mRNA level, the standard being taken from a second biological sampleobtained from an individual not having the disorder or being determinedby averaging levels from a population of individuals not having adisorder of the nervous system. As will be appreciated in the art, oncea standard Ependymin protein level or mRNA level is known, it can beused repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains Ependymin protein or mRNA. As indicated, biologicalsamples include body fluids (such as sera, plasma, urine, synovial fluidand spinal fluid) which contain free Ependymin protein, nervous systemtissue, and other tissue sources found to express complete or matureEpendymin or a Ependymin receptor. Methods for obtaining tissue biopsiesand body fluids from mammals are well known in the art. Where thebiological sample is to include mRNA, a tissue biopsy is the preferredsource.

The present invention is useful for diagnosis or treatment of variousnervous system-related disorders in mammals, preferably humans. Suchdisorders include any disregulations of nervous cell function including,but not limited to, Parkinson's disease, Alzheimer's disease,amyotrophic lateral sclerosis, pain, stroke, depression, anxiety,epilepsy, other neurological and psychiatric disorders, and the like.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described byChomczynski and Sacchi (Anal. Biochem. 162:156-159 (1987)). Levels ofmRNA encoding the Ependymin protein are then assayed using anyappropriate method. These include Northern blot analysis, S1 nucleasemapping, the polymerase chain reaction (PCR), reverse transcription incombination with the polymerase chain reaction (RT-PCR), and reversetranscription in combination with the ligase chain reaction (RT-LCR).

Assaying Ependymin protein levels in a biological sample can occur usingantibody-based techniques. For example, Ependymin protein expression intissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-basedmethods useful for detecting Ependymin protein gene expression includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andthe radioimmunoassay (RIA). Suitable antibody assay labels are known inthe art and include enzyme labels, such as, glucose oxidase, andradioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³¹S),tritium (³H), indium (¹¹² In), and technetium (^(99m)Tc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying Ependymin protein levels in a biological sampleobtained from an individual, Ependymin protein can also be detected invivo by imaging. Antibody labels or markers for in vivo imaging ofEpendymin protein include those detectable by X-radiography, NMR or ESR.For X-radiography, suitable labels include radioisotopes such as bariumor cesium, which emit detectable radiation but are not overtly harmfulto the subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

A Ependymin protein-specific antibody or antibody fragment which hasbeen labeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for immune systemdisorder. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ^(99m)Tc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain Ependymin protein. Invivo tumor imaging is described by Burchiel and coworkers (Chapter 13 inTumor Imaging: The Radiochemical Detection of Cancer, Burchiel, S. W.and Rhodes, B. A., eds., Masson Publishing Inc. (1982)).

Treatment

As noted above, Ependymin polynucleotides and polypeptides are usefulfor diagnosis of conditions involving abnormally high or low expressionof Ependymin activities. Given the cells and tissues where Ependymin isexpressed as well as the activities modulated by Ependymin, it isreadily apparent that a substantially altered (increased or decreased)level of expression of Ependymin in an individual compared to thestandard or “normal” level produces pathological conditions related tothe bodily system(s) in which Ependymin is expressed and/or is active.

It is well-known in the art that, in addition to a specific cellularfunction, cellular receptor molecules may also often be exploited by avirus as a means of initiating entry into a potential host cell. Forexample, it was recently discovered by Wu and colleagues (J. Exp. Med.185:1681-1691 (1997)) that the cellular chemokine receptor CCR5functions not only as a cellular chemokine receptor, but also as areceptor for macrophage-tropic human immunodeficiency virus (HIV)-1. Inaddition, RANTES, MIP-1a, and MIP-1b, which are agonists for thecellular chemokine receptor CCR5, inhibit entry of various strains ofHIV-1 into susceptible cell lines (Cocchi, F., et al., Science270:1811-1815 (1995)). Thus, the invention also provides a method oftreating an individual exposed to, or infected with, a virus through theprophylactic or therapeutic administration of Ependymin, or an agonistor antagonist thereof, to block or disrupt the interaction of a viralparticle with the Ependymin receptor and, as a result, block theinitiation or continuation of viral infectivity.

The Ependymin of the present invention binds to the Ependymin receptorand, as such, is likely to block neurotropic viral infections. Further,Ependymin expression is also observed in many bone and cartilagetissues, and, as such, Ependymin is also likely to block initiation ofinfectious cycle of many viruses which infect bone or cartilage. Morespecifically, a non-limiting list of viruses whose infectious lifecycles may be altered by Ependymin includes retroviruses such as HIV-1,HIV-2, human T-cell lymphotropic virus (HTLV)-I, and HTLV-II, as well asother DNA and RNA viruses including herpes simplex virus (HSV)-1, HSV-2,HSV-6, cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes samirii,adenoviruses, rhinoviruses, influenza viruses, reoviruses, and the like.

The ability of Ependymin of the present invention, or agonists orantagonists thereof, to prophylactically or therapeutically block viralinfection may be easily tested by the skilled artisan. For example,Simmons and coworkers (Science 276:276-279 (1997)) andArenzana-Seisdedos and colleagues (Nature 383:400 (1996)) each outline amethod of observing suppression of HIV-1 infection by an antagonist ofthe CCR5 chemokine receptor and of the CC chemokine RANTES,respectively, in cultured peripheral blood mononuclear cells. Cells arecultured and infected with a virus, HIV-1 in both cases noted above. Anagonist or antagonist of the CC chemokine or its receptor is thenimmediately added to the culture medium. Evidence of the ability of theagonist or antagonist of the chemokine or cellular receptor isdetermined by evaluating the relative success of viral infection at 3,6, and 9 days postinfection.

Administration of a pharmaceutical composition comprising an amount ofan isolated Ependymin, or an agonist or antagonist thereof, of theinvention to an individual either infected with a virus or at risk forinfection with a virus is performed as described below.

It will also be appreciated by one of ordinary skill that, since theEpendymin protein of the invention is a member of the ependyminpolypeptide family, the mature secreted form of the protein may bereleased in soluble form from the cells which express the Ependymin byproteolytic cleavage. Therefore, when Ependymin mature form is addedfrom an exogenous source to cells, tissues or the body of an individual,the protein will exert its physiological activities on its target cellsof that individual.

Therefore, it will be appreciated that conditions caused by a decreasein the standard or normal level of Ependymin activity in an individual,particularly disorders of the nervous system, can be treated byadministration of Ependymin polypeptide (in the form of the matureprotein). Thus, the invention also provides a method of treatment of anindividual in need of an increased level of Ependymin activitycomprising administering to such an individual a pharmaceuticalcomposition comprising an amount of an isolated Ependymin polypeptide ofthe invention, particularly a mature form of the Ependymin protein ofthe invention, effective to increase the Ependymin activity level insuch an individual.

Ependymin is found bound to collagen fibrils in the extracellular spaceof the mammalian leptomeninges. Ependymin, or agonists or antagoniststhereof, may thus be employed in the treatment of ependymitis or otherinflammation of the cellular membrane lining the central canal of thespinal cord and the brain ventricles. Ependymin is also found incollagen fibrils covering the endothelial cells of numerous bloodvessels. Similarly, Ependymin, or agonists or antagonists thereof, maybe used to regulate angiogenesis, and other processes or disorders whichinvolve the formation, maintenance or disorganization of blood vessels,and to treat a variety of cancers which involve the formation of newblood vessels. A list of such cancers may include, but is not limitedby, breast cancer, colon cancer, cardiac tumors, pancreatic cancer,melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, adenoma, and thelike.

Ependymins and other antiadhesive extracellular matrix proteins areinvolved in the mechanism whereby meningeal cells influence endfootformation of Bergmann glial cells, which organize the superficial glialimitans surrounding the CNS. This function is essential in establishingaccurate early development of the CNS and, as such, Ependymin of thepresent invention, or agonists or antagonists thereof, may be used totreat developmental disorders of the CNS and the brain. Ependymin mayalso be used to treat additional neurodegenerative disorders (such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Retinitis pigmentosa, Cerebellar degeneration, and the like. Further,Ependymin of the present invention, or agonists or antagonists thereof,may be used to modulate long-term memory consolidation.

Ependymin of the present invention, or agonists or antagonists thereof,may be used to enhance the regeneration of the optic or other nerves.Several scientific studies have revealed that goldfish ependyminexpression increases during optic nerve regeneration and thatanti-ependymin antibodies can prevent the sharpening of the regeneratingretinotectal projection (Schmidt, R. and Shashoua, V. E. J. Neurochem.36:1368-1377 (1988); Thomodsson, F. R., et al., Exp. Neurol. 117:260-268(1992)).

Ependymin of the present invention, or agonists or antagonists thereof,may be used to treat disorders resulting from axon ingrowth, forexample, along blood vessels or into the meninx, or prophylactically, toprevent undesired or incorrect neuronal growth.

Ependymin of the present invention, or agonists or antagonists thereof,may be used to treat disorders of the blood-brain barrier sinceependymin participates in the endothelial cell barrier by modulatingcell-matrix interactions.

Formulations

The Ependymin polypeptide composition will be formulated and dosed in afashion consistent with good medical practice, taking into account theclinical condition of the individual patient (especially the sideeffects of treatment with Ependymin polypeptide alone), the site ofdelivery of the Ependymin polypeptide composition, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” of Ependymin polypeptidefor purposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofEpendymin polypeptide administered parenterally per dose will be in therange of about 1 μg/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the Ependymin polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

Pharmaceutical compositions containing the Ependymin of the inventionmay be administered orally, rectally, parenterally, intracistemally,intravaginally, intraperitoneally, topically (as by powders, ointments,drops or transdermal patch), bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

The Ependymin polypeptide is also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or microcapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U.,et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethylmethacrylate; Langer, R., et al., J. Biomed. Mater. Res. 15:167-277(1981), and Langer, R., Chem. Tech. 12:98-105 (1982)), ethylene vinylacetate (Langer, R., et al., Id.) or poly-D-(−)-3-hydroxybutyric acid(EP 133,988). Sustained-release Ependymin polypeptide compositions alsoinclude liposomally entrapped Ependymin polypeptide. Liposomescontaining Ependymin polypeptide are prepared by methods known in theart (DE 3,218,121; Epstein, et al., Proc. Natl. Acad. Sci. (USA)82:3688-3692 (1985); Hwang, et al., Proc. Natl. Acad. Sci. (USA)77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324). Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal Ependymin polypeptide therapy.

For parenteral administration, in one embodiment, the Ependyminpolypeptide is formulated generally by mixing it at the desired degreeof purity, in a unit dosage injectable form (solution, suspension, oremulsion), with a pharmaceutically acceptable carrier, i.e., one that isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation. For example,the formulation preferably does not include oxidizing agents and othercompounds that are known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the Ependyminpolypeptide uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The Ependymin polypeptide is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of Ependymin polypeptide salts.

Ependymin polypeptide to be used for therapeutic administration must besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic Ependyminpolypeptide compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

Ependymin polypeptide ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous Ependymin polypeptide solution, andthe resulting mixture is lyophilized. The infusion solution is preparedby reconstituting the lyophilized Ependymin polypeptide usingbacteriostatic water-for-injection (WFI).

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention may be employed in conjunctionwith other therapeutic compounds.

Agonists and Antagonists—Assays and Molecules

The invention also provides a method of screening compounds to identifythose which enhance or block the action of Ependymin on cells, such asits interaction with Ependymin-binding molecules such as receptormolecules. An agonist is a compound which increases the naturalbiological functions of Ependymin or which functions in a manner similarto Ependymin, while antagonists decrease or eliminate such functions.

In another aspect of this embodiment the invention provides a method foridentifying a receptor protein or other ligand-binding protein whichbinds specifically to a Ependymin polypeptide. For example, a cellularcompartment, such as a membrane or a preparation thereof, may beprepared from a cell that expresses a molecule that binds Ependymin. Thepreparation is incubated with labeled Ependymin and complexes ofEpendymin bound to the receptor or other binding protein are isolatedand characterized according to routine methods known in the art.Alternatively, the Ependymin polypeptide may be bound to a solid supportso that binding molecules solubilized from cells are bound to the columnand then eluted and characterized according to routine methods.

In the assay of the invention for agonists or antagonists, a cellularcompartment, such as a membrane or a preparation thereof, may beprepared from a cell that expresses a molecule that binds Ependymin,such as a molecule of a signaling or regulatory pathway modulated byEpendymin. The preparation is incubated with labeled Ependymin in theabsence or the presence of a candidate molecule which may be a Ependyminagonist or antagonist. The ability of the candidate molecule to bind thebinding molecule is reflected in decreased binding of the labeledligand. Molecules which bind gratuitously, i.e., without inducing theeffects of Ependymin on binding the Ependymin binding molecule, are mostlikely to be good antagonists. Molecules that bind well and eliciteffects that are the same as or closely related to Ependymin areagonists.

Ependymin-like effects of potential agonists and antagonists may bymeasured, for instance, by determining activity of a second messengersystem following interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that ofEpendymin or molecules that elicit the same effects as Ependymin. Secondmessenger systems that may be useful in this regard include but are notlimited to AMP guanylate cyclase, ion channel or phosphoinositidehydrolysis second messenger systems.

Another example of an assay for Ependymin antagonists is a competitiveassay that combines Ependymin and a potential antagonist withmembrane-bound Ependymin receptor molecules or recombinant Ependyminreceptor molecules under appropriate conditions for a competitiveinhibition assay. Ependymin can be labeled, such as by radioactivity,such that the number of Ependymin molecules bound to a receptor moleculecan be determined accurately to assess the effectiveness of thepotential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducingEpendymin-induced activities, thereby preventing the action of Ependyminby excluding Ependymin from binding.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through triple-helix formation. Antisense techniques arediscussed in a number of studies (for example, Okano, J. Neurochem.56:560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression.” CRC Press, Boca Raton, Fla. (1988)). Triple helix formationis discussed in a number of studies, as well (for instance, Lee, et al.,Nucleic Acids Research 6:3073 (1979); Cooney, et al., Science 241:456(1988); Dervan, et al., Science 251:1360 (1991)). The methods are basedon binding of a polynucleotide to a complementary DNA or RNA. Forexample, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of Ependymin. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into Ependymin polypeptide. The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of Ependyminprotein.

The agonists and antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described above.

The antagonists may be employed for instance to inhibit the formation ofEpendymin-collagen fibrils which typically cover the endothelial cellsof numerous blood vessels. As a result, anti-Ependymin antibodies may beused to regulate angiogenesis, and other processes or disorders whichinvolve the formation, maintenance or disorganization of blood vessels,and to treat a variety of cancers which involve the formation of newblood vessels. Antibodies against Ependymin may also be employed to bindto and inhibit Ependymin activity to treat Parkinson's disease,Alzheimer's disease, amyotrophic lateral sclerosis, pain, stroke,depression, anxiety, epilepsy, and other neurological and psychiatricdisorders. Any of the above antagonists may be employed in a compositionwith a pharmaceutically acceptable carrier, e.g., as hereinafterdescribed.

Gene Mapping

The nucleic acid molecules of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a Ependymin protein gene. Thiscan be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthen is used for in situ chromosome mapping using well known techniquesfor this purpose.

In addition, in some cases, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Fluorescence in situ hybridization (“FISH”) of a cDNA cloneto a metaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp (for a review of this technique,see Verma, et al., Human Chromosomes: A Manual Of Basic Techniques,Pergamon Press, New York (1988)).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, on the WorldWide Web (McKusick, V. Mendelian Inheritance In Man, available on-linethrough Johns Hopkins University, Welch Medical Library). Therelationship between genes and diseases that have been mapped to thesame chromosomal region are then identified through linkage analysis(coinheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1(a) Expression and Purification of “His-Tagged”Ependymin in E. coli

The novel pHE4 series of bacterial expression vectors, in particular,the pHE4-5 vector may be used for bacterial expression in this example.pHE4-5/MPIFD23 vector plasmid DNA contains an insert which encodesanother ORF. The construct was deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, onSep. 30, 1997 and given Accession No. 209311. Using the Nde I and Asp718 restriction sites flanking the irrelevant MPIF ORF insert, thepHE4-5 is linearized and the MPIF ORF is removed.

The pHE4-5 bacterial expression vector includes a neomycinphosphotransferase gene for selection, an E. coli origin of replication,a T5 phage promoter sequence, two lac operator sequences, aShine-Dalgarno sequence, and the lactose operon repressor gene (lacIq).These elements are arranged such that an inserted DNA fragment encodinga polypeptide expresses that polypeptide with the six His residues(i.e., a “6×His tag”) covalently linked to the amino terminus of thatpolypeptide. The promoter and operator sequences of the pHE4-5 vectorwere made synthetically. Synthetic production of nucleic acid sequencesis well known in the art (CLONETECH 95/96 Catalog, pages 215-216,CLONETECH, 1020 East Meadow Circle, Palo Alto, Calif. 94303).

The DNA sequence encoding the desired portion of the Ependymin proteincomprising the mature form of the Ependymin amino acid sequence isamplified from the deposited cDNA clone using PCR oligonucleotideprimers which anneal to the amino terminal sequences of the desiredportion of the Ependymin protein and to sequences in the depositedconstruct 3′ to the cDNA coding sequence. Additional nucleotidescontaining restriction sites to facilitate cloning in the pHE4-5 vectorare added to the 5′ and 3′ primer sequences, respectively.

For cloning the mature form of the Ependymin protein, the 5′ primer hasthe sequence 5′ GCG CAT ATG GCC CCG CGC CCG TGC 3′ (SEQ ID NO:8)containing the underlined Nde I restriction site followed by 15nucleotides of the amino terminal coding sequence of the matureEpendymin sequence in SEQ ID NO:2. One of ordinary skill in the artwould appreciate, of course, that the point in the protein codingsequence where the 5′ primer begins may be varied to amplify a DNAsegment encoding any desired portion of the complete Ependymin proteinshorter or longer than the mature form of the protein. The 3′ primer hasthe sequence 5′ GCG GGT ACC TCA CCA GGA GCA GTC TTC GC 3′ (SEQ ID NO:9)containing the underlined Asp 718 restriction site followed by 20nucleotides complementary to the 3′ end of the coding sequence of theEpendymin DNA sequence in FIGS. 1A, 1B, and 1C.

The amplified Ependymin DNA fragment and the vector pHE4-5 are digestedwith Nde I and Asp 718 and the digested DNAs are then ligated together.Insertion of the Ependymin DNA into the restricted pHE4-5 vector placesthe Ependymin protein coding region downstream from the IPTG-induciblepromoter and in-frame with an initiating AUG and the six histidinecodons.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures such as those described by Sambrook and colleagues(Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance (“Kanr”),is used in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressingEpendymin protein, is available commercially (QIAGEN, Inc., supra).Transformants are identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-β-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

The cells are then stirred for 3-4 hours at 4° C. in 6M guanidine-HCl,pH 8. The cell debris is removed by centrifugation, and the supernatantcontaining the Ependymin is loaded onto a nickel-nitrilo-tri-acetic acid(“Ni-NTA”) affinity resin column (QIAGEN, Inc., supra). Proteins with a6×His tag bind to the Ni-NTA resin with high affinity and can bepurified in a simple one-step procedure (for details see: TheQIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly the supernatant isloaded onto the column in 6 M guanidine-HCl, pH 8, the column is firstwashed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10volumes of 6 M guanidine-HCl pH 6, and finally the Ependymin is elutedwith 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins can be eluted by the addition of 250 mMimidazole. Imidazole is removed by a final dialyzing step against PBS or50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified proteinis stored at 4° C. or frozen at −80° C.

The following alternative method may be used to purify Ependyminexpressed in E coli when it is present in the form of inclusion bodies.Unless otherwise specified, all of the following steps are conducted at4-10° C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10° C. and the cells are harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells ware then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the Ependyminpolypeptide-containing supernatant is incubated at 4° C. overnight toallow further GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4° C. without mixing for 12 hours prior tofurther purification steps.

To clarify the refolded Ependymin polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 μm membranefilter with appropriate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 mm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

Fractions containing the Ependymin polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the Ependymin polypeptide (determined, for instance, by 16%SDS-PAGE) are then pooled.

The resultant Ependymin polypeptide exhibits greater than 95% purityafter the above refolding and purification steps. No major contaminantbands are observed from Commassie blue stained 16% SDS-PAGE gel when 5μg of purified protein is loaded. The purified protein is also testedfor endotoxin/LPS contamination, and typically the LPS content is lessthan 0.1 ng/ml according to LAL assays.

Example 2 Cloning and Expression of Ependymin Protein in a BaculovirusExpression System

In this illustrative example, the plasmid shuttle vector pA2 is used toinsert the cloned DNA encoding complete protein, including its naturallyassociated secretory signal (leader) sequence, into a baculovirus toexpress the mature Ependymin protein, using standard methods asdescribed by Summers and colleagues (A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures, Texas AgriculturalExperimental Station Bulletin No. 1555 (1987)). This expression vectorcontains the strong polyhedrin promoter of the Autographa californicanuclear polyhedrosis virus (AcMNPV) followed by convenient restrictionsites such as Bam HI, Xba I and Asp 718. The polyadenylation site of thesimian virus 40 (“SV40”) is used for efficient polyadenylation. For easyselection of recombinant virus, the plasmid contains thebeta-galactosidase gene from E. coli under control of a weak Drosophilapromoter in the same orientation, followed by the polyadenylation signalof the polyhedrin gene. The inserted genes are flanked on both sides byviral sequences for cell-mediated homologous recombination withwild-type viral DNA to generate a viable virus that express the clonedpolynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, by Luckow andcoworkers (Virology 170:31-39 (1989)).

The cDNA sequence encoding the full length Ependymin protein in thedeposited clone, including the AUG initiation codon and the naturallyassociated leader sequence shown in SEQ ID NO:2, is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene. The 5′ primer has the sequence 5′ GAT CGC TCT AGA TCC GCC ACC ATGCCA GGA CGC GCT CCC CTC CGC ACC GTC 3′ (SEQ ID NO:10) containing theunderlined Xba I restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.196:947-950 (1987)), followed by 27 of nucleotides of the sequence ofthe complete Ependymin protein shown in FIGS. 1A, 1B, and 1C, beginningwith the AUG initiation codon. The 3′ primer has the sequence 5′ GAT CGCGGT ACC TTA TCA CCA GGA GCA GTC TTC GCT CAT CTT CTC CAG 3′ (SEQ IDNO:11) containing the underlined Asp 718 restriction site followed by 30nucleotides complementary to the 3′ noncoding sequence in FIGS. 1A, 1B,and 1C.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with Xba I and Asp 718 and againis purified on a 1% agarose gel. This fragment is designated herein“V1”.

The plasmid is digested with the restriction enzymes Xba I and Asp 718and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vector DNA isdesignated herein “V1”.

Fragment F1 and the dephosphorylated plasmid V1 are ligated togetherwith T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts suchas XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria are identified that contain the plasmid with the humanEpendymin gene by digesting DNA from individual colonies using Xba I andAsp 718 and then analyzing the digestion product by gel electrophoresis.The sequence of the cloned fragment is confirmed by DNA sequencing. Thisplasmid is designated herein pA2Ependymin.

Five μg of the plasmid pA2Ependymin is co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner and colleagues (Proc. Natl. Acad. Sci. USA84:7413-7417 (1987)). One μg of BaculoGold™ virus DNA and 5 μg of theplasmid pA2Ependymin are mixed in a sterile well of a microtiter platecontaining 50 μl of serum-free Grace's medium (Life Technologies Inc.,Frederick, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace's mediumare added, mixed and incubated for 15 minutes at room temperature. Thenthe transfection mixture is added drop-wise to Sf9 insect cells (ATCCCRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace'smedium without serum. The plate is then incubated for 5 hours at 27° C.The transfection solution is then removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum is added.Cultivation is then continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith (supra). An agarose gelwith “Blue Gal” (Life Technologies Inc., Frederick, Md.) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Frederick, Md., page 9-10). After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Therecombinant virus is called V-Ependymin.

To verify the expression of the Ependymin gene Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus V-Ependymin at a multiplicityof infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6hours later the medium is removed and is replaced with SF900 II mediumminus methionine and cysteine (available from Life Technologies Inc.,Frederick, Md.). After 42 hours, 5 μCi of ³⁵S-methionine and 5 μCi³⁵S-cysteine (available from Amersham) are added. The cells are furtherincubated for 16 hours and then are harvested by centrifugation. Theproteins in the supernatant as well as the intracellular proteins areanalyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe mature form of the Ependymin protein and thus the cleavage point andlength of the naturally associated secretory signal peptide.

Example 3 Cloning and Expression of Ependymin in Mammalian Cells

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, hygromycin allows theidentification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulto develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS; Murphy, et al., Biochem J.227:277-279 (1991); Bebbington, et al., Bio/Technology 10: 169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of proteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen, et al., Mol. Cel. Biol. 5:438-447(1985)) plus a fragment of the CMV-enhancer (Boshart, et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites Bam HI, Xba I and Asp 718, facilitate the cloningof the gene of interest. The vectors contain in addition the 3′ intron,the polyadenylation and termination signal of the rat preproinsulingene.

Example 3(a) Cloning and Expression in COS Cells

The expression plasmid, pEpendyminHA, is made by cloning a portion ofthe cDNA encoding the complete Ependymin protein into the expressionvector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen,Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson and colleagues (Cell 37:767 (1984)). The fusion of the HA tag tothe target protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

A DNA fragment encoding the complete Ependymin polypeptide is clonedinto the polylinker region of the vector so that recombinant proteinexpression is directed by the CMV promoter. The plasmid constructionstrategy is as follows. The Ependymin cDNA of the deposited clone isamplified using primers that contain convenient restriction sites, muchas described above for construction of vectors for expression ofEpendymin in E. coli. Suitable primers include the following, which areused in this example. The 5′ primer, containing the underlined Asp 718site, a Kozak sequence, an AUG start codon, a sequence, and 24nucleotides of the 5′ coding region of the complete Ependyminpolypeptide, has the following sequence: 5′ GAT CGC GGT ACC GCC ATC ATGCCA GGA CGC GCT CCC CTC CGC 3′ (SEQ ID NO:12). The 3′ primer, containingthe underlined Bam HI and 20 of nucleotides complementary to the 3′coding sequence immediately before the stop codon, has the followingsequence: 5′ GAT CGC GGA TCC TCA CCA GGA GCA GTC TTC GC 3′ (SEQ IDNO:13).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith Asp 718 and Bam HI and then ligated. The ligation mixture istransformed into E. coli strain SURE (Stratagene Cloning Systems, LaJolla, Calif. 92037), and the transformed culture is plated onampicillin media plates which then are incubated to allow growth ofampicillin resistant colonies. Plasmid DNA is isolated from resistantcolonies and examined by restriction analysis or other means for thepresence of the fragment encoding the complete Ependymin polypeptide.

For expression of recombinant Ependymin, COS cells are transfected withan expression vector, as described above, using DEAE-dextran, asdescribed, for instance, by Sambrook and coworkers (Molecular Cloning: aLaboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor,N.Y. (1989)). Cells are incubated under conditions for expression ofEpendymin by the vector.

Expression of the Ependymin-HA fusion protein is detected byradiolabeling and immunoprecipitation, using methods described in, forexample Harlow and colleagues (Antibodies: A Laboratory Manual, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)).To this end, two days after transfection, the cells are labeled byincubation in media containing ³⁵S-cysteine for 8 hours. The cells andthe media are collected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson andcolleagues (supra). Proteins are precipitated from the cell lysate andfrom the culture media using an HA-specific monoclonal antibody. Theprecipitated proteins then are analyzed by SDS-PAGE and autoradiography.An expression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 3(b) Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of Ependymin polypeptide.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.37146). The plasmid contains the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Life Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W., etal., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C.Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. andSydenham, M. A. Biotechnology 9:64-68 (1991)). Cells grown in increasingconcentrations of MTX develop resistance to the drug by overproducingthe target enzyme, DHFR, as a result of amplification of the DHFR gene.If a second gene is linked to the DHFR gene, it is usually co-amplifiedand over-expressed. It is known in the art that this approach may beused to develop cell lines carrying more than 1,000 copies of theamplified gene(s). Subsequently, when the methotrexate is withdrawn,cell lines are obtained which contain the amplified gene integrated intoone or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Mol. Cell. Biol. 5:438-447 (1985)) plus a fragmentisolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV; Boshart, et al., Cell 41:521-530 (1985)).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: Bam HI, Xba I,and Asp 718. Behind these cloning sites the plasmid contains the 3′intron and polyadenylation site of the rat preproinsulin gene. Otherhigh efficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the Ependymin polypeptide in a regulatedway in mammalian cells (Gossen, M., and Bujard, H. Proc. Natl. Acad.Sci. USA 89:5547-5551 (1992)). For the polyadenylation of the mRNA othersignals, e.g., from the human growth hormone or globin genes can be usedas well. Stable cell lines carrying a gene of interest integrated intothe chromosomes can also be selected upon co-transfection with aselectable marker such as gpt, G418 or hygromycin. It is advantageous touse more than one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC4 is digested with the restriction enzymes Xba I and Asp718 and then dephosphorylated using calf intestinal phosphates byprocedures known in the art. The vector is then isolated from a 1%agarose gel.

The DNA sequence encoding the complete Ependymin polypeptide isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ sequences of the desired portion of the gene. The 5′ primercontaining the underlined Xba I site, a Kozak sequence (in italics), anAUG start codon, and 30 nucleotides of the 5′ coding region of thecomplete Ependymin polypeptide, has the following sequence: 5′ GAT CGCTCT AGA TCC GCC ACC ATG CCA GGA CGC GCT CCC CTC CGC ACC GTC 3′ (SEQ IDNO:14). The 3′ primer, containing the underlined Asp 718 and 30 ofnucleotides complementary to the 3′ coding sequence immediately beforethe stop codon as shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1), has thesequence shown in the above example as SEQ ID NO:11.

The amplified fragment is digested with the endonucleases Xba I and Asp718 and then purified again on a 1% agarose gel. The isolated fragmentand the dephosphorylated vector are then ligated with T4 DNA ligase. E.coli HB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using lipofectin (Felgner, et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 4 Tissue Distribution of Ependymin mRNA Expression

Northern blot analysis is carried out to examine Ependymin geneexpression in human tissues, using methods described by, among others,Sambrook and colleagues (supra). A cDNA probe containing the entirenucleotide sequence of the Ependymin protein (SEQ ID NO:1) is labeledwith ³²P using the rediprime™ DNA labeling system (Amersham LifeScience), according to manufacturer's instructions. After labeling, theprobe is purified using a CHROMA SPIN-100™ column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labeled probe is then used to examine varioushuman tissues for Ependymin mRNA.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) are obtained from Clontech andare examined with the labeled probe using ExpressHyb™ hybridizationsolution (Clontech) according to manufacturer's protocol numberPT1190-1. Following hybridization and washing, the blots are mounted andexposed to film at −70° C. overnight, and films developed according tostandard procedures.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

Further, the Sequence Listing submitted herewith, and the SequenceListing submitted with U.S. application Ser. No. 09/229,583, filed onJan. 13, 1999, the Sequence Listing submitted with U.S. ProvisionalApplication Ser. No. 60/071,330, filed on Jan. 14, 1998, and theSequence Listing submitted with U.S. Provisional Application Ser. No.60/075,278, filed on Feb. 19, 1998 (to both of which the presentapplication claims benefit of the filing date under 35 U.S.C. § 119(e)),in both computer and paper forms are hereby incorporated by reference intheir entireties.

1. An isolated antibody or fragment thereof that specifically binds toan Ependymin polypeptide selected from the group consisting of: (a) anEpendymin polypeptide whose amino acid sequence consists of amino acidresidues −37 to +187 of SEQ ID NO:2; (b) an Ependymin polypeptide whoseamino acid sequence consists of amino acid residues 1 to +187 of SEQ IDNO:2; (c) an Ependymin polypeptide whose amino acid sequence consists ofthe full-length polypeptide encoded by the cDNA in ATCC Deposit No.209464; and (d) an Ependymin polypeptide whose amino acid sequenceconsists of the mature polypeptide encoded by the cDNA in ATCC DepositNo.
 209464. 2. The antibody or fragment thereof of claim 1 thatspecifically binds the Ependymin polypeptide of (a).
 3. The antibody orfragment thereof of claim 1 that specifically binds the Ependyminpolypeptide of (b).
 4. The antibody or fragment thereof of claim 1 thatspecifically binds the Ependymin polypeptide of (c).
 5. The antibody orfragment thereof of claim 1 that specifically binds the Ependyminpolypeptide of (d).
 6. The antibody or fragment thereof of claim 1wherein said antibody or fragment thereof is monoclonal.
 7. The antibodyor fragment thereof of claim 1 wherein said antibody or fragment thereofis polyclonal or chimeric.
 8. The antibody or fragment thereof of claim1 wherein said antibody or fragment thereof is a Fab fragment.
 9. Theantibody or fragment thereof of claim 1 which is labeled.
 10. Theantibody or fragment thereof of claim 1 wherein said antibody orfragment thereof specifically binds to said polypeptide in a Westernblot or ELISA.
 11. A hybridoma that produces the antibody or fragmentthereof of claim
 1. 12. A method of detecting an Ependymin polypeptidein a biological sample comprising: (a) contacting the biological samplewith the antibody or fragment thereof of claim 2; (b) allowing a complexto form between said Ependymin polypeptide and said antibody; and (c)detecting said complex.
 13. An isolated antibody or fragment thereofthat specifically binds to an Ependymin polypeptide selected from thegroup consisting of: (a) a polypeptide whose amino acid sequenceconsists of a fragment of SEQ ID NO:2, wherein said fragment is at least30 contiguous amino acid residues of SEQ lID NO:2 in length; (b) apolypeptide whose amino acid sequence consists of a fragment of SEQ ID)NO:2, wherein said fragment is at least 50 contiguous amino acidresidues of SEQ ID NO:2 in length; (c) a polypeptide whose amino acidsequence consists of a fragment of the Ependymin polypeptide encoded bythe cDNA contained in ATCC Deposit No. 209464, wherein said fragment isat least 30 contiguous amino acid residues in length; and (d) apolypeptide whose amino acid sequence consists of a fragment of theEpendymin polypeptide encoded by the cDNA in ATCC Deposit No. 209464,wherein said fragment is at least 50 contiguous amino acid residues inlength.
 14. The antibody or fragment thereof of claim 13 wherein saidantibody or fragment thereof is monoclonal.
 15. The antibody or fragmentthereof of claim 13 wherein said antibody or fragment thereof ispolyclonal or chimenc.
 16. The antibody or fragment thereof of claim 13which is a Fab fragment.
 17. The antibody or fragment thereof of claim13 wherein said antibody or fragment thereof specifically binds to saidprotein in a Westem blot or ELISA.
 18. An isolated antibody or fragmentthereof that specifically binds a Ependymin protein purified from a cellculture wherein said protein is encoded by a polynucleotide encodingamino acids −37 to +187 of SEQ ID NO:2.
 19. The antibody or fragmentthereof of claim 18 wherein said antibody or fragment thereof ismonoclonal.
 20. The antibody or fragment thereof of claim 18 whereinsaid antibody or fragment thereof is polyclonal or chimeric.
 21. Theantibody or fragment thereof of claim 18 which is a Fab fragment. 22.The antibody or fragment thereof of claim 13 wherein said antibody orfragment thereof specifically binds to said protein in a Western blot orELISA.